Thiophene analogues for the treatment or prevention of flavivirus infections

ABSTRACT

Compounds represented by formula I: 
                         
or pharmaceutically acceptable salts and solvates thereof, wherein R 1 , X, Y, and Z are as defined herein, are useful for treating flaviviridae viral infections.

This application claims the benefit of U.S. Provisional PatentApplication No. 60/858,939, filed Nov. 15, 2006, the entire disclosureof which is hereby incorporated by reference.

The present invention relates to novel compounds and a method for thetreatment or prevention of Flavivirus infections using novel compounds.

Hepatitis is a disease occurring throughout the world. It is generallyof viral nature, although there are other causes known. Viral hepatitisis by far the most common form of hepatitis. Nearly 750,000 Americansare affected by hepatitis each year, and out of those, more than 150,000are infected with the hepatitis C virus (“HCV”).

HCV is a positive-stranded RNA virus belonging to the Flaviviridaefamily and has closest relationship to the pestiviruses that include hogcholera virus and bovine viral diarrhea virus (BVDV). HCV is believed toreplicate through the production of a complementary negative-strand RNAtemplate. Due to the lack of efficient culture replication system forthe virus, HCV particles were isolated from pooled human plasma andshown, by electron microscopy, to have a diameter of about 50-60 nm. TheHCV genome is a single-stranded, positive-sense RNA of about 9,600 bpcoding for a polyprotein of 3009-3030 amino-acids, which is cleaved coand post-translationally into mature viral proteins (core, E1, E2, p7,NS2, NS3, NS4A, NS4B, NS5A, NS5B). It is believed that the structuralglycoproteins, E1 and E2, are embedded into a viral lipid envelope andform stable heterodimers. It is also believed that the structural coreprotein interacts with the viral RNA genome to form the nucleocapsid.The nonstructural proteins designated NS2 to NS5 include proteins withenzymatic functions involved in virus replication and protein processingincluding a polymerase, protease and helicase.

The main source of contamination with HCV is blood. The magnitude of theHCV infection as a health problem is illustrated by the prevalence amonghigh-risk groups. For example, 60% to 90% of hemophiliacs and more than80% of intravenous drug abusers in western countries are chronicallyinfected with HCV. For intravenous drug abusers, the prevalence variesfrom about 28% to 70% depending on the population studied. Theproportion of new HCV infections associated with post-transfusion hasbeen markedly reduced lately due to advances in diagnostic tools used toscreen blood donors.

Combination of pegylated interferon plus ribavirin is the treatment ofchoice for chronic HCV infection. This treatment does not providesustained viral response (SVR) in a majority of patients infected withthe most prevalent genotype (1a and 1b). Furthermore, significant sideeffects prevent compliance to the current regimen and may require dosereduction or discontinuation in some patients.

There is therefore a great need for the development of anti-viral agentsfor use in treating or preventing Flavivirus infections.

In one aspect, the present invention provides a compound of formula I:

or pharmaceutically acceptable salts thereof;wherein,

-   R¹ is C₁₋₆ alkyl, C₃₋₆ cycloalkyl, or C₆₋₁₄ aryl which is    substituted one or more times by —NH(C₁₋₄ alkyl), —N(C₁₋₄ alkyl)₂,    hydroxyl, NH₂, 3-12 member heterocycle, or NHSO₂C₆₋₁₈ aryl;-   Z is H, halogen, C₁₋₆ alkyl which is unsubstituted or substituted    one or more times by R¹⁰, C₂₋₆ alkenyl which is unsubstituted or    substituted one or more times by R¹⁰, or C₂₋₆ alkynyl which is    unsubstituted or substituted one or more times by R¹⁰;-   X is

-   M is

-   R⁵ is cyclohexyl which is unsubstituted or substituted one or more    times by R¹³;-   R⁶ is

or cyclohexyl which is substituted one or more times by R¹⁴;

-   Y is COOR⁷, COCOOR⁷, P(O)OR^(a)OR^(b), S(O)OR⁷, S(O)₂OR⁷, tetrazole,    CON(R⁷)CH(R⁷)COOR⁷, CONR⁸R⁹, CON(R⁷)—SO₂—R⁷, CONR⁷OH and halogen;-   R⁷, R⁸ and R⁹ are each independently H, C₁₋₁₂ alkyl which is    unsubstituted or substituted one or more times by R¹⁰, C₂₋₁₂ alkenyl    which is unsubstituted or substituted one or more times by R¹⁰,    C₂₋₁₂ alkynyl which is unsubstituted or substituted one or more    times by R¹⁰, C₆₋₁₄ aryl which is unsubstituted or substituted one    or more times by R¹¹, C₇₋₁₆ aralkyl which is unsubstituted or    substituted one or more times by R¹¹, 5-12 member heteroaryl which    is unsubstituted or substituted one or more times by R¹¹, 6-18    member heteroaralkyl which is unsubstituted or substituted one or    more times by R¹¹, 3-12 member heterocycle which is unsubstituted or    substituted one or more times by R¹², or 4-18 member    heterocycle-alkyl which is unsubstituted or substituted one or more    times by R¹², or R⁸ and R⁹ are taken together with the nitrogen atom    to form a 3 to 10 member heterocycle which is unsubstituted or    substituted one or more times by R¹² or a 5-12 member heteroaryl    which is unsubstituted or substituted one or more times by R¹¹; and-   R_(a) and R_(b) are each independently chosen from H, C₁₋₁₂ alkyl    which is unsubstituted or substituted one or more times by R¹⁰,    C₂₋₁₂ alkenyl which is unsubstituted or substituted one or more    times by R¹⁰, C₂₋₁₂ alkynyl which is unsubstituted or substituted    one or more times by R¹⁰, C₆₋₁₄ aryl which is unsubstituted or    substituted one or more times by R¹¹, C₇₋₁₆ aralkyl which is    unsubstituted or substituted one or more times by R¹¹, 5-12 member    heteroaryl which is unsubstituted or substituted one or more times    by R¹¹, 6-18 member heteroaralkyl which is unsubstituted or    substituted one or more times by R¹¹, 3-12 member heterocycle which    is unsubstituted or substituted one or more times by R¹², or 4-18    member heterocycle-alkyl which is unsubstituted or substituted one    or more times by R¹², or R^(a) and R^(b) are taken together with the    oxygen atoms to form a 5 to 10 member heterocycle which is    unsubstituted or substituted one or more times by R¹⁰ or a 5-12    member heteroaryl which is unsubstituted or substituted one or more    times by R¹¹;-   R¹⁰ is halogen, oxo, —NH₂, —NH(C₁₋₄ alkyl), —N(C₁₋₄ alkyl)₂, —CONH₂,    —CONH(C₁₋₄ alkyl), —CON(C₁₋₄ alkyl)₂, —NHCOH, —N(C₁₋₄ alkyl)COH,    —N(C₁₋₄ alkyl)COC₁₋₄ alkyl, —NHCOC₁₋₄ alkyl, —C(O)H, —C(O)C₁₋₄    alkyl, carboxy, —C(O)OC₁₋₄ alkyl, hydroxyl, C₁₋₄ alkoxy, nitro,    nitroso, azido, cyano, —S(O)₀₋₂H, —S(O)₀₋₂C₁₋₄ alkyl, —SO₂NH₂,    —SO₂NH(C₁₋₄ alkyl), —SO₂N(C₁₋₄ alkyl)₂, —NHSO₂H, —N(C₁₋₄ alkyl)SO₂H,    —N(C₁₋₄ alkyl)SO₂C₁₋₄ alkyl, or —NHSO₂C₁₋₄ alkyl;-   R¹¹ is halogen, C₁₋₆ alkyl, halogenated C₁₋₆ alkyl, C₂₋₆ alkenyl,    C₂₋₆ alkynyl, C₁₋₆ alkoxy, —NH₂, —NH(C₁₋₄ alkyl), —N(C₁₋₄ alkyl)₂,    —CONH₂, —CONH(C₁₋₄ alkyl), —CON(C₁₋₄ alkyl)₂, —NHCOH, —N(C₁₋₄    alkyl)COH, —N(C₁₋₄ alkyl)COC₁₋₄ alkyl, —NHCOC₁₋₄ alkyl, —C(O)H,    —C(O)C₁₋₄ alkyl, carboxy, —C(O)OC₁₋₄ alkyl, hydroxyl, C₁₋₆ alkoxy,    nitro, nitroso, azido, cyano, —S(O)₀₋₂H, —S(O)₀₋₂C₁₋₄ alkyl,    —SO₂NH₂, —SO₂NH(C₁₋₄ alkyl), —SO₂N(C₁₋₄ alkyl)₂, —NHSO₂H, —N(C₁₋₄    alkyl)SO₂H, —N(C₁₋₄ alkyl)SO₂C₁₋₄ alkyl, or —NHSO₂C₁₋₄ alkyl;-   R¹² is halogen, oxo, C₁₋₆ alkyl, halogenated C₁₋₆ alkyl, C₂₋₆    alkenyl, C₂₋₆ alkynyl, —NH₂, —NH(C₁₋₄ alkyl), —N(C₁₋₄ alkyl)₂,    —CONH₂, —CONH(C₁₋₄ alkyl), —CON(C₁₋₄ alkyl)₂, —NHCOH, —N(C₁₋₄    alkyl)COH, —N(C₁₋₄ alkyl)COC₁₋₄ alkyl, —NHCOC₁₋₄ alkyl, —C(O)H,    —C(O)C₁₋₄ alkyl, carboxy, —C(O)OC₁₋₄ alkyl, hydroxyl, C₁₋₆ alkoxy,    nitro, nitroso, azido, cyano, —S(O)₀₋₂H, —S(O)₀₋₂C₁₋₄ alkyl,    —SO₂NH₂, —SO₂NH(C₁₋₄ alkyl), —SO₂N(C₁₋₄ alkyl)₂, —NHSO₂H, —N(C₁₋₄    alkyl)SO₂H, —N(C₁₋₄ alkyl)SO₂C₁₋₄ alkyl, or —NHSO₂C₁₋₄ alkyl;-   R¹³ is OH, halogen, C₁₋₆-alkyl, halogenated C₁₋₆-alkyl, C₁₋₆-alkoxy,    halogenated C₁₋₆-alkoxy, cyano, nitro, —NH₂, —NH(C₁₋₄ alkyl),    —N(C₁₋₄ alkyl)₂, —CONH₂, —CONH(C₁₋₄ alkyl), —CON(C₁₋₄ alkyl)₂,    —NHCOH, —N(C₁₋₄ alkyl)COH, —N(C₁₋₄ alkyl)COC₁₋₄ alkyl, —NHCOC₁₋₄    alkyl, —C(O)H, —C(O)C₁₋₄ alkyl, carboxy, —C(O)OC₁₋₄ alkyl,    —S(O)₀₋₂C₁₋₄ alkyl, —SO₂NH₂, —SO₂NH(C₁₋₄ alkyl), —SO₂N(C₁₋₄ alkyl)₂,    —N(C₁₋₄ alkyl)SO₂H, —N(C₁₋₄ alkyl)SO₂C₁₋₄ alkyl, —NHSO₂C₁₋₄ alkyl,    C₆₋₁₄ aryl, C₆₋₁₄ aryloxy, or C₆₋₁₄-aryloxy-C₁₋₆-alkyl;-   R¹⁴ is OH, halogen, C₁₋₆-alkoxy, C₁₋₆-alkyl, C₁₋₆-alkyl-CO—NH—,    C₁₋₆-alkyl-CO—N(C₁₋₆-alkyl)-, or 5 to 10 member heteroaryl; and-   R^(14a) is C₁₋₆-alkyl, C₃₋₇-cycloalkyl, halogenated C₁₋₆-alkyl,    C₁₋₆-alkyl-CO—, —S(O)₀₋₂C₁₋₄ alkyl, 5 to 10 member heteroaryl or    C₆₋₁₄-aryl.

The compounds of the present invention are HCV polymerase inhibitors.Surprisingly, it has been found that the compounds according to thepresent invention and having a specific substitution pattern, exhibitimproved properties relative to other thiophene HCV polymeraseinhibitors. It is therefore believed that the compounds of the presentinvention have excellent potential for the treatment and prevention ofhepatitis C infections.

In another aspect, there is provided a method for treating or preventinga Flaviviridae viral infection in a patient comprising administering tothe patient a therapeutically effective amount of a compound,composition or combination of the invention.

In another aspect, there is provided a pharmaceutical compositioncomprising at least one compound of the invention and at least onepharmaceutically acceptable carrier or excipient.

In another aspect, there is provided a combination comprising a compoundof the invention and one or more additional agents chosen from viralserine protease inhibitors, viral polymerase inhibitors, viral helicaseinhibitors, immunomudulating agents, antioxidant agents, antibacterialagents, therapeutic vaccines, hepatoprotectant agents, antisense agent,inhibitors of HCV NS2/3 protease and inhibitors of internal ribosomeentry site (IRES).

In a further aspect, there is provided the use of a compound,composition or combination of the invention for treating or preventing aFlaviviridae viral infection in a patient.

In still another aspect, there is provided the use of a compound,composition or combination of the invention for inhibiting or reducingthe activity of viral polymerase in a patient.

In still another aspect, there is provided the use of a compound,composition or combination of the invention for the manufacture of amedicament for treating or preventing a viral Flaviviridae infection ina patient.

In one embodiment, compounds of the present invention comprise thosewherein the following embodiments are present, either independently orin combination.

In accordance with a preferred compound or method aspect, the compoundsof the present invention are represented by formula IA:

or pharmaceutically acceptable salts thereof;wherein, each of X, Y and R¹ are as defined above.

In accordance with a further preferred compound or method aspect, thecompounds of the present invention are represented by formula IB:

or pharmaceutically acceptable salts thereof;wherein each of X, Y, R¹, R⁵, and R⁶ are as defined above.

According to a further embodiment, R¹ in formulas I, IA, and IB is C₁₋₆alkyl or C₃₋₆cycloalkyl which are unsubstituted or substituted one ormore times by —NH₂, NHCH₃, N(CH₃)₂, or hydroxyl.

According to a further embodiment, R¹ in formulas I, IA, and IB is C₁₋₆alkyl which is unsubstituted or substituted one or more times by—NH₂NHCH₃, N(CH₃)₂, or hydroxyl.

According to a further embodiment, R¹ in formulas I, IA, and IB is C₁₋₆alkyl.

According to a further embodiment, R¹ in formulas I, IA, and IB is R¹ ismethyl, ethyl, propyl, isopropyl, butyl, sec.-butyl, or tert.-butyl.

According to a further embodiment, R¹ in formulas I, IA, and IB iscyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

According to a further embodiment, R¹ in formulas I, IA, and IB isphenyl

According to a further embodiment, Z in formula I is H, halogen, or C₁₋₆alkyl which is unsubstituted or substituted one or more times by R¹⁰.

According to a further embodiment, Z in formula I is H, halogen, or C₁₋₄alkyl which is unsubstituted or substituted one or more times by R¹⁰.

According to a further embodiment, Z in formula I is H or C₁₋₄ alkyl.

According to a further embodiment, Z in formula I is H or methyl.

According to a further embodiment, Y in formulas I, IA, and IB, isCOOR⁷, and R⁷ is H, C₁₋₁₂ alkyl which is unsubstituted or substitutedone or more times by R¹⁰, C₂₋₁₂ alkenyl which is unsubstituted orsubstituted one or more times by R¹⁰, C₂₋₁₂ alkynyl which isunsubstituted or substituted one or more times by R¹⁰, or C₆₋₁₄ arylwhich is unsubstituted or substituted one or more times by R¹¹.

According to a further embodiment, Y in formulas I, IA, and IB, isCOOR⁷, and R⁷ is H, C₁₋₆ alkyl which is unsubstituted or substituted oneor more times by R¹⁰, or C₆₋₁₄ aryl which is unsubstituted orsubstituted one or more times by R¹¹.

According to a further embodiment, Y in formulas I, IA, and IB, isCOOR⁷, and R⁷ is H, C₁₋₄ alkyl which is unsubstituted or substituted oneor more times by R¹⁰, or phenyl which is unsubstituted or substitutedone or more times by R¹¹.

According to a further embodiment, Y in formulas I, IA, and IB, is COOR⁷and R⁷ is H, C₁₋₄ alkyl or phenyl.

According to a further embodiment, Y in formulas I, IA, and IB, is COOR⁷and R⁷ is H, methyl, or ethyl.

According to a further embodiment, Y in formulas I, IA, and IB, is COOR⁷and R⁷ is H.

According to further embodiment, R⁵ in formulas I, IA, and IB iscyclohexyl which is unsubstituted or substituted one or more times byOH, halogen, C₁₋₆-alkyl, halogenated C₁₋₆-alkyl, C₁₋₆-alkoxy,halogenated C₁₋₆-alkoxy, cyano, nitro, —NH₂, —NH(C₁₋₄ alkyl), or —N(C₁₋₄alkyl)₂.

According to further embodiment, R⁵ in formulas I, IA, and IB iscyclohexyl which is substituted in the 4-position.

According to further embodiment, in formulas I, IA, and IB, X is—NR⁶—CO—R⁵ and R⁵ is cyclohexyl which is substituted in the 4-positionand the 4-position substituent is in the trans position relative to thecarbonyl.

According to further embodiment, R⁵ in formulas I, IA, and IB iscyclohexyl which is unsubstituted or substituted in the 4-position byOH, halogen, C₁₋₆-alkyl, halogenated C₁₋₆-alkyl, C₁₋₆-alkoxy,halogenated C₁₋₆-alkoxy, cyano, nitro, —NH₂, —NH(C₁₋₄ alkyl), or —N(C₁₋₄alkyl)₂.

According to further embodiment, in formulas I, IA, and IB, X is—NR⁶—CO—R⁵ and R⁵ is cyclohexyl which is unsubstituted or substituted inthe 4-position by OH, halogen, C₁₋₆-alkyl, halogenated C₁₋₆-alkyl,C₁₋₆-alkoxy, halogenated C₁₋₆-alkoxy, cyano, nitro, —NH₂, —NH(C₁₋₄alkyl), or —N(C₁₋₄ alkyl)₂, wherein the 4-position substituent is in thetrans position relative to the carbonyl group.

According to further embodiment, R⁶ in formulas I, IA, and IB is R⁶ iscyclohexyl which is substituted one or more times by OH, halogen,C₁₋₆-alkyl, or C₁₋₆-alkoxy.

According to further embodiment, R⁶ in formulas I, IA, and IB iscyclohexyl which is substituted in the 4-position.

According to further embodiment, R⁶ in formulas I, IA, and IB iscyclohexyl which is substituted in the 4-position and the 4-positionsubstituent is in the trans position relative to the amino group.

According to further embodiment, R⁶ in formulas I, IA, and IB iscyclohexyl which is substituted in the 4-position by OH or C₁₋₆-alkoxy.

According to further embodiment, R⁶ in formulas I, IA, and IB is

and R^(14a) is C₁₋₆-alkyl, halogenated C₁₋₆-alkyl, C₁₋₆-alkyl-CO—,—S(O)₀₋₂C₁₋₄ alkyl, heteroaryl or C₆₋₁₄-aryl.

According to further embodiment, R⁶ in formulas I, IA, and IB is

and R^(14a) is C₁₋₆-alkyl, halogenated C₁₋₆-alkyl, C₁₋₆-alkyl-CO—,—S(O)₀₋₂C₁₋₄ alkyl, heteroaryl or C₆₋₁₄-aryl.

According to further embodiment, R⁶ in formulas I, IA, and IB is

and R^(14a) is methyl, ethyl, propyl or isopropyl.

According to further embodiment, in formulas I, or IB, R⁶ is cyclohexylwhich is substituted in the 4-position by OH, C₁₋₆-alkyl, or C₁₋₆-alkoxywherein the 4-position substituent is in the trans position relative tothe amino group.

According to further embodiment, in formulas I, or IB, R⁶ is cyclohexylwhich is substituted in the 4-position by OH, C₁₋₆-alkyl, or C₁₋₆-alkoxywherein the 4-position substituent is in the trans position relative tothe amino group, and R⁵ is cyclohexyl which is unsubstituted orsubstituted in the 4-position by OH, halogen, C₁₋₆-alkyl, halogenatedC₁₋₆-alkyl, C₁₋₆-alkoxy, halogenated C₁₋₆-alkoxy, cyano, nitro, —NH₂,—NH(C₁₋₄ alkyl), or —N(C₁₋₄ alkyl)₂, wherein the 4-position substituentis in the trans position relative to the carbonyl group.

In accordance with a preferred embodiment of the invention, thecompounds of the present invention are selected from the compounds offormula (IB) and pharmaceutically acceptable salts thereof, wherein:

-   -   R¹ is C₁₋₆ alkyl or C₃₋₆ cycloalkyl which in each case is        unsubstituted or substituted one or more times by —NH₂, NHCH₃,        N(CH₃)₂, or hydroxyl;    -   R⁵ is cyclohexyl which is unsubstituted or substituted one or        more times by OH, C₁₋₄-alkyl, and/or C₁₋₄-alkoxy;    -   R⁶ is cyclohexyl which is substituted one or more times by OH,        Hal (e.g., F), C₁₋₄-alkoxy, C₁₋₄-alkyl, C₁₋₄-alkyl-CO—NH—,        C₁₋₄-alkyl-CO—N(C₁₋₄-alkyl)-, or triazolyl;    -   Y is COOR⁷; and    -   R⁷ is H, C₁₋₁₂ alkyl or C₆₋₁₄ aryl.

In accordance with a preferred embodiment of the invention, thecompounds of the present invention are selected from the compounds offormula (IB) and pharmaceutically acceptable salts thereof, wherein:

-   -   R¹ is C₁₋₆ alkyl or C₃₋₆ cycloalkyl;    -   R⁵ is cyclohexyl which is unsubstituted or substituted one or        more times by OH, C₁₋₄-alkyl, and/or C₁₋₄-alkoxy;    -   R⁶ is cyclohexyl which is substituted one or more times by OH,        Hal (e.g., F), C₁₋₄-alkoxy, C₁₋₄-alkyl, C₁₋₄-alkyl-CO—NH—,        C₁₋₄-alkyl-CO—N(C₁₋₄-alkyl)-, or triazolyl;    -   Y is COOR⁷; and    -   R⁷ is H, C₁₋₁₂ alkyl or C₆₋₁₄ aryl.

In accordance with another preferred embodiment of the invention, thecompounds of the present invention are selected from the compounds offormula (IB) and pharmaceutically acceptable salts thereof, wherein:

-   -   R¹ is C₁₋₆ alkyl which is unsubstituted or substituted one or        more times by —NH₂, NHCH₃, N(CH₃)₂, or hydroxyl;    -   R⁵ is cyclohexyl which is unsubstituted or substituted in the        4-position by OH, C₁₋₄-alkyl, and/or C₁₋₄-alkoxy;    -   R⁶ is cyclohexyl which is substituted in the 4-position by OH,        Hal (e.g., F), C₁₋₄-alkoxy, C₁₋₄-alkyl, C₁₋₄-alkyl-CO—NH—,        C₁₋₄-alkyl-CO—N(C₁₋₄-alkyl)-, or triazolyl;    -   Y is COOR⁷; and    -   R⁷ is H, C₁₋₁₂ alkyl or C₆₋₁₄ aryl.

In accordance with another preferred embodiment of the invention, thecompounds of the present invention are selected from the compounds offormula (IB) and pharmaceutically acceptable salts thereof, wherein:

-   -   R¹ is C₁₋₆ alkyl which is unsubstituted or substituted one or        more times by —NH₂, NHCH₃, N(CH₃)₂, or hydroxyl;    -   R⁵ is cyclohexyl which is unsubstituted or substituted in the        4-position by OH, C₁₋₄-alkyl, and/or C₁₋₄-alkoxy;    -   R⁶ is cyclohexyl which is substituted in the 4-position by OH,        Hal (e.g., F), C₁₋₄-alkoxy, C₁₋₄-alkyl, C₁₋₄-alkyl-CO—NH—,        C₁₋₄-alkyl-CO—N(C₁₋₄-alkyl)-, or triazolyl;    -   Y is COOR⁷; and    -   R⁷ is H, C₁₋₁₂ alkyl or C₆₋₁₄ aryl.

In accordance with another preferred embodiment of the invention, thecompounds of the present invention are selected from the compounds offormula (IB) and pharmaceutically acceptable salts thereof, wherein:

-   -   R¹ is C₁₋₆ alkyl which is unsubstituted or substituted one or        more times by —NH₂, NHCH₃, N(CH₃)₂, or hydroxyl;    -   R⁵ is cyclohexyl which is unsubstituted or substituted one or        more times by OH, C₁₋₄-alkyl, or C₁₋₄-alkoxy;    -   R⁶ is cyclohexyl which is substituted one or more times by OH,        F, C₁₋₄-alkoxy, C₁₋₄-alkyl, or halogenated C₁₋₄-alkyl;    -   Y is COOR⁷; and    -   R⁷ is H or C₁₋₄ alkyl.        In accordance with another preferred embodiment of the        invention, the compounds of the present invention are selected        from the compounds of formula (IB) and pharmaceutically        acceptable salts thereof, wherein:    -   R¹ is C₁₋₆ alkyl;    -   R⁵ is cyclohexyl which is unsubstituted or substituted one or        more times by OH, C₁₋₄-alkyl, or C₁₋₄-alkoxy;    -   R⁶ is cyclohexyl which is substituted one or more times by OH,        F, C₁₋₄-alkoxy, or C₁₋₄-alkyl;    -   Y is COOR⁷; and    -   R⁷ is H or C₁₋₄ alkyl.

In accordance with another preferred embodiment of the invention, thecompounds of the present invention are selected from the compounds offormula (IB) and pharmaceutically acceptable salts thereof, wherein:

-   -   R¹ is C₁₋₆ alkyl or C₃₋₆ cycloalkyl which in each case is        unsubstituted or substituted one or more times by —NH₂, NHCH₃,        N(CH₃)₂, or hydroxyl;    -   R⁵ is cyclohexyl which is unsubstituted or substituted one or        more times by OH, C₁₋₄-alkyl, or C₁₋₄-alkoxy;    -   R⁶ is cyclohexyl which is substituted one or more times by OH,        F, C₁₋₄-alkoxy, or C₁₋₄-alkyl;    -   Y is COOR⁷; and    -   R⁷ is H or C₁₋₄ alkyl.

In accordance with another preferred embodiment of the invention, thecompounds of the present invention are selected from the compounds offormula (IB) and pharmaceutically acceptable salts thereof, wherein:

-   -   R¹ is C₁₋₆ alkyl or C₃₋₆ cycloalkyl;    -   R⁵ is cyclohexyl which is unsubstituted or substituted one or        more times by OH, C₁₋₄-alkyl, or C₁₋₄-alkoxy;    -   R⁶ is cyclohexyl which is substituted one or more times by OH,        F, C₁₋₄-alkoxy, or C₁₋₄-alkyl;    -   Y is COOR⁷; and    -   R⁷ is H or C₁₋₄ alkyl.

In accordance with another preferred embodiment of the invention, thecompounds of the present invention are selected from the compounds offormula (IB) and pharmaceutically acceptable salts thereof, wherein:

-   -   R¹ is methyl, ethyl, propyl, isopropyl, butyl, sec.-butyl,        tert.-butyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl        which in each case is unsubstituted or substituted one or more        times by —NH₂, NHCH₃, N(CH₃)₂, or hydroxyl;    -   R⁵ is cyclohexyl which is unsubstituted or substituted one or        more times by OH, C₁₋₄-alkyl, or C₁₋₄-alkoxy;    -   R⁶ is cyclohexyl which is substituted one or more times by OH,        F, C₁₋₄-alkoxy, C₁₋₄-alkyl, or halogenated C₁₋₄-alkyl;    -   Y is COOR⁷; and    -   R⁷ is H or C₁₋₄ alkyl.

In accordance with another preferred embodiment of the invention, thecompounds of the present invention are selected from the compounds offormula (IB) and pharmaceutically acceptable salts thereof, wherein:

-   -   R¹ is methyl, ethyl, propyl, isopropyl, butyl, sec.-butyl,        tert.-butyl, cyclopropyl, cyclobutyl, cyclopentyl, or        cyclohexyl;    -   R⁵ is cyclohexyl which is unsubstituted or substituted one or        more times by OH, C₁₋₄-alkyl, or C₁₋₄-alkoxy;    -   R⁶ is cyclohexyl which is substituted one or more times by OH,        F, C₁₋₄-alkoxy, C₁₋₄-alkyl, or halogenated C₁₋₄-alkyl;    -   Y is COOR⁷; and    -   R⁷ is H or C₁₋₄ alkyl.

In accordance with another preferred embodiment of the invention, thecompounds of the present invention are selected from the compounds offormula (IB) and pharmaceutically acceptable salts thereof, wherein:

-   -   R¹ is methyl, ethyl, propyl, isopropyl, butyl, sec.-butyl,        tert.-butyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl        which in each case is unsubstituted or substituted one or more        times by —NH₂, NHCH₃, N(CH₃)₂, or hydroxyl —NH₂, NHCH₃, N(CH₃)₂,        or hydroxyl;    -   R⁵ is cyclohexyl which is unsubstituted or substituted one or        more times by OH, C₁₋₄-alkyl, or C₁₋₄-alkoxy;    -   R⁶ is cyclohexyl which is substituted one or more times by OH,        F, C₁₋₄-alkoxy, C₁₋₄-alkyl, or halogenated C₁₋₄-alkyl;    -   Y is COOH.

In accordance with another preferred embodiment of the invention, thecompounds of the present invention are selected from the compounds offormula (IB) and pharmaceutically acceptable salts thereof, wherein:

-   -   R¹ is methyl, ethyl, propyl, isopropyl, butyl, sec.-butyl,        tert.-butyl, cyclopropyl, cyclobutyl, cyclopentyl, or        cyclohexyl;    -   R⁵ is cyclohexyl which is unsubstituted or substituted one or        more times by OH, C₁₋₄-alkyl, or C₁₋₄-alkoxy;    -   R⁶ is cyclohexyl which is substituted one or more times by OH,        F, C₁₋₄-alkoxy, C₁₋₄-alkyl, or halogenated C₁₋₄-alkyl;    -   Y is COOH.

In accordance with another preferred embodiment of the invention, thecompounds of the present invention are selected from the compounds offormula (IB) and pharmaceutically acceptable salts thereof, wherein:

-   -   R¹ is C₁₋₆ alkyl or C₃₋₆ cycloalkyl which in each case is        unsubstituted or substituted one or more times by —NH₂, NHCH₃,        N(CH₃)₂, or hydroxyl;    -   R⁵ is cyclohexyl which is unsubstituted or substituted one or        more times by OH, C₁₋₄-alkyl, and/or C₁₋₄-alkoxy;    -   R⁶ is

-   -   R^(14a) is C₁₋₆-alkyl, halogenated C₁₋₆-alkyl, C₁₋₆-alkyl-CO—,        —S(O)₀₋₂C₁₋₄ alkyl, heteroaryl or C₆₋₁₄-aryl;    -   Y is COOR⁷; and    -   R⁷ is H, C1-12 alkyl or C6-14 aryl.        In accordance with another preferred embodiment of the        invention, the compounds of the present invention are selected        from the compounds of formula (IB) and pharmaceutically        acceptable salts thereof, wherein:    -   R¹ is C₁₋₆ alkyl or C₃₋₆ cycloalkyl;    -   R⁵ is cyclohexyl which is unsubstituted or substituted one or        more times by OH, C₁₋₄-alkyl, and/or C₁₋₄-alkoxy;    -   R⁶ is

-   -   R^(14a) is C₁₋₆-alkyl, halogenated C₁₋₆-alkyl, C₁₋₆-alkyl-CO—,        —S(O)₀₋₂C₁₋₄ alkyl, heteroaryl or C₆₋₁₄-aryl;    -   Y is COOR⁷; and    -   R⁷ is H, C₁₋₁₂ alkyl or C₆₋₁₄ aryl.

In accordance with another preferred embodiment of the invention, thecompounds of the present invention are selected from the compounds offormula (IB) and pharmaceutically acceptable salts thereof, wherein:

-   -   R¹ is C₁₋₆ alkyl or C₃₋₆ cycloalkyl which in each case is        unsubstituted or substituted one or more times by —NH₂, NHCH₃,        N(CH₃)₂, or hydroxyl;    -   R⁵ is cyclohexyl which is unsubstituted or substituted one or        more times by OH, C₁₋₄-alkyl, and/or C₁₋₄-alkoxy;    -   R⁶ is

-   -   R^(14a) is C₁₋₆-alkyl or halogenated C₁₋₆-alkyl;    -   Y is COOR⁷; and    -   R⁷ is H, C₁₋₁₂ alkyl or C₆₋₁₄ aryl.

In accordance with another preferred embodiment of the invention, thecompounds of the present invention are selected from the compounds offormula (IB) and pharmaceutically acceptable salts thereof, wherein:

-   -   R¹ is C₁₋₆ alkyl or C₃₋₆ cycloalkyl;    -   R⁵ is cyclohexyl which is unsubstituted or substituted one or        more times by OH, C₁₋₄-alkyl, and/or C₁₋₄-alkoxy;    -   R⁶ is

-   -   R^(14a) is C₁₋₆-alkyl or halogenated C₁₋₆-alkyl;    -   Y is COOR⁷; and    -   R⁷ is H, C₁₋₁₂ alkyl or C₆₋₁₄ aryl.

In accordance with another preferred embodiment of the invention, thecompounds of the present invention are selected from the compounds offormula (IB) and pharmaceutically acceptable salts thereof, wherein:

-   -   R¹ is C₁₋₆ alkyl or C₃₋₆ cycloalkyl which in each case is        unsubstituted or substituted one or more times by —NH₂, NHCH₃,        N(CH₃)₂, or hydroxyl;    -   R⁵ is cyclohexyl which is unsubstituted or substituted one or        more times by OH, C₁₋₄-alkyl, and/or C₁₋₄-alkoxy;    -   R⁶ is

-   -   R^(14a) is C₁₋₆-alkyl;    -   Y is COOR⁷; and    -   R⁷ is H, C₁₋₁₂ alkyl or C₆₋₁₄ aryl.

In accordance with another preferred embodiment of the invention, thecompounds of the present invention are selected from the compounds offormula (IB) and pharmaceutically acceptable salts thereof, wherein:

-   -   R¹ is C₁₋₆ alkyl or C₃₋₆ cycloalkyl;    -   R⁵ is cyclohexyl which is unsubstituted or substituted one or        more times by OH, C₁₋₄-alkyl, and/or C₁₋₄-alkoxy;    -   R⁶ is

-   -   R^(14a) is C₁₋₆-alkyl;    -   Y is COOR⁷; and    -   R⁷ is H, C₁₋₁₂ alkyl or C₆₋₁₄ aryl.

In accordance with another preferred embodiment of the invention, thecompounds of the present invention are selected from the compounds offormula (IB) and pharmaceutically acceptable salts thereof, wherein:

-   -   R¹ is C₁₋₆ alkyl or C₃₋₆ cycloalkyl which in each case is        unsubstituted or substituted one or more times by —NH₂, NHCH₃,        N(CH₃)₂, or hydroxyl;    -   R⁵ is cyclohexyl which is unsubstituted or substituted one or        more times by OH, C₁₋₄-alkyl, and/or C₁₋₄-alkoxy;    -   R⁶ is

-   -   R^(14a) is methyl, ethyl, propyl, isopropyl, butyl, sec.-butyl,        or tert.-butyl;    -   Y is COOR⁷; and    -   R⁷ is H, C₁₋₁₂ alkyl or C₆₋₁₄ aryl.

In accordance with another preferred embodiment of the invention, thecompounds of the present invention are selected from the compounds offormula (IB) and pharmaceutically acceptable salts thereof, wherein:

-   -   R¹ is C₁₋₆ alkyl or C₃₋₆ cycloalkyl;    -   R⁵ is cyclohexyl which is unsubstituted or substituted one or        more times by OH, C₁₋₄-alkyl, and/or C₁₋₄-alkoxy;    -   R⁶ is

-   -   R^(14a) is methyl, ethyl, propyl, isopropyl, butyl, sec.-butyl,        or tert.-butyl;    -   Y is COOR⁷; and    -   R⁷ is H, C₁₋₁₂ alkyl or C₆₋₁₄ aryl.

According to an aspect of the invention, the compounds of the inventionare selected from:

-   5-(3,3-DIMETHYL-BUT-1-YNYL)-3-[(TRANS-4-HYDROXY-CYCLOHEXYL)-(TRANS-4-METHYL-CYCLOHEXANECARBONYL)-AMINO]-THIOPHENE-2-CARBOXYLIC    ACID;-   5-(3,3-DIMETHYL-BUT-1-YNYL)-3-[(TRANS-4-METHOXY-CYCLOHEXYL)-(TRANS-4-METHYL-CYCLOHEXANECARBONYL)-AMINO]-THIOPHENE-2-CARBOXYLIC    ACID;-   5-(3,3-DIMETHYL-BUT-1-YNYL)-3-[(TRANS-4-METHYL-CYCLOHEXANECARBONYL)-(CIS-4-[1,2,4]TRIAZOL-1-YL-CYCLOHEXYL)-AMINO]-THIOPHENE-2-CARBOXYLIC    ACID;-   5-(3,3-DIMETHYL-BUT-1-YNYL)-3-[(TRANS-4-METHYL-CYCLOHEXANECARBONYL)-(TRANS-4-[1,2,4]TRIAZOL-1-YL-CYCLOHEXYL)-AMINO]-THIOPHENE-2-CARBOXYLIC    ACID;-   5-(3,3-DIMETHYL-BUT-1-YNYL)-3-[(CIS-4-HYDROXY-CYCLOHEXYL)-(TRANS-4-METHYL-CYCLOHEXANECARBONYL)-AMINO]-THIOPHENE-2-CARBOXYLIC    ACID;-   5-(3,3-DIMETHYL-BUT-1-YNYL)-3-[(TRANS-4-METHYL-CYCLOHEXANECARBONYL)-(1-METHYL-PIPERIDIN-4-YL)-AMINO]-THIOPHENE-2-CARBOXYLIC    ACID; HYDROCHLORIDE;-   5-(3,3-DIMETHYL-BUT-1-YNYL)-3-[(TRANS-4-METHYL-CYCLOHEXANECARBONYL)-(4-CIS-[1,2,3]TRIAZOL-1-YL-CYCLOHEXYL)-AMINO]-THIOPHENE-2-CARBOXYLIC    ACID-   5-(3,3-DIMETHYL-BUT-1-YNYL)-3-[(TRANS-4-METHYL-CYCLOHEXANECARBONYL)-(TRANS-4-[1,2,3]TRIAZOL-1-YL-CYCLOHEXYL)-AMINO]-THIOPHENE-2-CARBOXYLIC    ACID; and-   5-(3,3-DIMETHYL-BUT-1-YNYL)-3-[(TRANS-4-FLUORO-CYCLOHEXYL)-(TRANS-4-METHYL-CYCLOHEXANECARBONYL)-AMINO]-THIOPHENE-2-CARBOXYLIC    ACID.

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising at least one compound according to the inventiondescribed herein and at least one pharmaceutically acceptable carrier orexcipient.

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising at least one compound according to the inventiondescribed herein and at least one compound according to the inventiondescribed herein, and further comprising administering at least oneadditional agent chosen from viral serine protease inhibitors, viralpolymerase inhibitors, viral helicase inhibitors, immunomudulatingagents, antioxidant agents, antibacterial agents, therapeutic vaccines,hepatoprotectant agents, antisense agents, inhibitors of HCV NS2/3protease and inhibitors of internal ribosome entry site (IRES).

In another embodiment, there is provided a combination comprising aleast one compound according to the invention described herein and oneor more additional agents chosen from viral serine protease inhibitors,viral polymerase inhibitors, viral helicase inhibitors, immunomudulatingagents, antioxidant agents, antibacterial agents, therapeutic vaccines,hepatoprotectant agents, antisense agent, inhibitors of HCV NS2/3protease and inhibitors of internal ribosome entry site (IRES).

In one combination embodiment, the compound and additional agent areadministered sequentially.

In another combination embodiment, the compound and additional agent areadministered simultaneously.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical formulation and thus pharmaceuticalformulations comprising a combination as defined above together with apharmaceutically acceptable carrier therefore comprise a further aspectof the invention.

The additional agents for the compositions and combinations include, forexample, ribavirin, amantadine, merimepodib, Levovirin, Viramidine, andmaxamine.

The term “viral serine protease inhibitor” as used herein means an agentthat is effective to inhibit the function of the viral serine proteaseincluding HCV serine protease in a mammal. Inhibitors of HCV serineprotease include, for example, those compounds described in WO 99/07733(Boehringer Ingelheim), WO 99/07734 (Boehringer Ingelheim), WO 00/09558(Boehringer Ingelheim), WO 00/09543 (Boehringer Ingelheim), WO 00/59929(Boehringer Ingelheim), WO 02/060926 (BMS), WO 2006039488 (Vertex), WO2005077969 (Vertex), WO 2005035525 (Vertex), WO 2005028502 (Vertex) WO2005007681 (Vertex), WO 2004092162 (Vertex), WO 2004092161 (Vertex), WO2003035060 (Vertex), of WO 03/087092 (Vertex), WO 02/18369 (Vertex), orWO98/17679 (Vertex).

The term “viral polymerase inhibitors” as used herein means an agentthat is effective to inhibit the function of a viral polymeraseincluding an HCV polymerase in a mammal. Inhibitors of HCV polymeraseinclude non-nucleosides, for example, those compounds described in:

WO 03/010140 (Boehringer Ingelheim), WO 03/026587 (Bristol MyersSquibb); WO 02/100846 A1, WO 02/100851 A2, WO 01/85172 A1 (GSK), WO02/098424 A1 (GSK), WO 00/06529 (Merck), WO 02/06246 A1 (Merck), WO01/47883 (Japan Tobacco), WO 03/000254 (Japan Tobacco) and EP 1 256 628A2 (Agouron).

Furthermore other inhibitors of HCV polymerase also include nucleosideanalogs, for example, those compounds described in: WO 01/90121 A2(Idenix), WO 02/069903 A2 (Biocryst Pharmaceuticals Inc.), and WO02/057287 A2(Merck/Isis) and WO 02/057425 A2 (Merck/Isis).

Specific examples of nucleoside inhibitors of an HCV polymerase, includeR1626/R1479 (Roche), R7128 (Roche), MK-0608 (Merck), R1656,(Roche-Pharmasset) and Valopicitabine (Idenix).

Specific examples of inhibitors of an HCV polymerase, includeJTK-002/003 and JTK-109 (Japan Tobacco), HCV-796 (Viropharma), GS-9190(Gilead), and PF-868,554 (Pfizer).

The term “viral helicase inhibitors” as used herein means an agent thatis effective to inhibit the function of a viral helicase including aFlaviviridae helicase in a mammal.

“Immunomodulatory agent” as used herein means those agents that areeffective to enhance or potentiate the immune system response in amammal. Immunomodulatory agents include, for example, class Iinterferons (such as α-, β-, δ- and Ω-interferons, τ-interferons,consensus interferons and asialo-interferons), class II interferons(such as γ-interferons) and pegylated interferons.

The term “class I interferon” as used herein means an interferonselected from a group of interferons that all bind to receptor type 1.This includes both naturally and synthetically produced class Iinterferons. Examples of class I interferons include α-, β-, δ- andΩ-interferons, τ-interferons, consensus interferons andasialo-interferons. The term “class II interferon” as used herein meansan interferon selected from a group of interferons that all bind toreceptor type II. Examples of class II interferons includeγ-interferons.

Antisense agents include, for example, ISIS-14803.

Specific examples of inhibitors of HCV NS3 protease, include BILN-2061(Boehringer Ingelheim) SCH-6 and SCH-503034/Boceprevir(Schering-Plough),VX-950/telaprevir (Vertex) and ITMN-B (InterMune), GS9132 (Gilead),TMC-435350 (Tibotec/Medivir), ITMN-191 (InterMune), MK-7009 (Merck).

Inhibitor internal ribosome entry site (IRES) include ISIS-14803 (ISISPharmaceuticals) and those compounds described in WO 2006019831 (PTCtherapeutics).

In one embodiment, the additional agent is interferon α, ribavirin,silybum marianum, interleukine-12, amantadine, ribozyme, thymosin,N-acetyl cysteine or cyclosporin.

In one embodiment, the additional agent is interferon α1A, interferonα1B, interferon α2A, or interferon α2B.

Interferon is available in pegylated and non pegylated forms. Pegylatedinterferons include PEGASYS™ and Peg-intron™.

The recommended dose of PEGASYS™ monotherapy for chronic hepatitis C is180 mg (1.0 mL vial or 0.5 mL prefilled syringe) once weekly for 48weeks by subcutaneous administration in the abdomen or thigh.

The recommended dose of PEGASYS™ when used in combination with ribavirinfor chronic hepatitis C is 180 mg (1.0 mL vial or 0.5 mL prefilledsyringe) once weekly.

The daily dose of Ribavirin is 800 mg to 1200 mg administered orally intwo divided doses. The dose should be individualized to the patientdepending on baseline disease characteristics (e.g., genotype), responseto therapy, and tolerability of the regimen.

The recommended dose of PEG-Intron™ regimen is 1.0 mg/kg/weeksubcutaneously for one year. The dose should be administered on the sameday of the week.

When administered in combination with ribavirin, the recommended dose ofPEG-Intron is 1.5 micrograms/kg/week.

In one embodiment, viral serine protease inhibitor is a flaviviridaeserine protease inhibitor.

In one embodiment, viral polymerase inhibitor is a flaviviridaepolymerase inhibitor.

In one embodiment, viral helicase inhibitor is a flaviviridae helicaseinhibitor.

In further embodiments:

viral serine protease inhibitor is HCV serine protease inhibitor;

viral polymerase inhibitor is HCV polymerase inhibitor;

viral helicase inhibitor is HCV helicase inhibitor.

In one embodiment, the present invention provides a method for treatingor preventing a Flaviviridae viral infection in a host comprisingadministering to the host a therapeutically effective amount of at leastone compound according to formula I.

In one embodiment, the viral infection is chosen from Flavivirusinfections.

In one embodiment, the Flavivirus infection is Hepatitis C virus (HCV),bovine viral diarrhea virus (BVDV), hog cholera virus, dengue fevervirus, Japanese encephalitis virus or yellow fever virus.

In one embodiment, the Flaviviridae viral infection is hepatitis C viralinfection (HCV).

In one embodiment, the present invention provides a method for treatingor preventing a Flaviviridae viral infection in a host comprisingadministering to the host a therapeutically effective amount of at leastone compound according to the invention described herein, and furthercomprising administering at least one additional agent chosen from viralserine protease inhibitors, viral polymerase inhibitors, viral helicaseinhibitors, immunomudulating agents, antioxidant agents, antibacterialagents, therapeutic vaccines, hepatoprotectant agents, antisense agents,inhibitors of HCV NS2/3 protease and inhibitors of internal ribosomeentry site (IRES).

In one embodiment, there is provided a method for inhibiting or reducingthe activity of viral polymerase in a host comprising administering atherapeutically effective amount of a compound according to theinvention described herein.

In one embodiment, there is provided a method for inhibiting or reducingthe activity of viral polymerase in a host comprising administering atherapeutically effective amount of a compound according to theinvention described herein and further comprising administering one ormore viral polymerase inhibitors.

In one embodiment, viral polymerase is a Flaviviridae viral polymerase.

In one embodiment, viral polymerase is a RNA-dependant RNA-polymerase.

In one embodiment, viral polymerase is HCV polymerase.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical formulation and thus pharmaceuticalformulations comprising a combination as defined above together with apharmaceutically acceptable carrier therefore comprise a further aspectof the invention.

The individual components for use in the method of the present inventionor combinations of the present invention may be administered eithersequentially or simultaneously in separate or combined pharmaceuticalformulations.

In one embodiment, the present invention provides the use of a compoundaccording to the invention described herein for treating or preventingFlaviviridae viral infection in a host.

In one embodiment, the present invention provides the use of a compoundaccording to the invention described herein for the manufacture of amedicament for treating or preventing a viral Flaviviridae infection ina host.

In one embodiment, the present invention provides the use of a compoundaccording to the invention described herein for inhibiting or reducingthe activity of viral polymerase in a host.

It will be appreciated by those skilled in the art that the compounds inaccordance with the present invention can exists as stereoisomers (forexample, optical (+ and −), geometrical (cis and trans) andconformational isomers (axial and equatorial). All such stereoisomersare included in the scope of the present invention.

It will be appreciated by those skilled in the art that the compounds inaccordance with the present invention can contain a chiral center. Thecompounds of formula may thus exist in the form of two different opticalisomers (i.e. (+) or (−) enantiomers). All such enantiomers and mixturesthereof including racemic mixtures are included within the scope of theinvention. The single optical isomer or enantiomer can be obtained bymethod well known in the art, such as chiral HPLC, enzymatic resolutionand chiral auxiliary.

In one embodiment, the compounds of the present invention are providedin the form of a single enantiomer at least 95%, at least 97% and atleast 99% free of the corresponding enantiomer.

In a further embodiment the compound of the present invention are in theform of the (+) enantiomer at least 95% free of the corresponding (−)enantiomer.

In a further embodiment the compound of the present invention are in theform of the (+) enantiomer at least 97% free of the corresponding (−)enantiomer.

In a further embodiment the compound of the present invention are in theform of the (+) enantiomer at least 99% free of the corresponding (−)enantiomer.

In a further embodiment, the compounds of the present invention are inthe form of the (−) enantiomer at least 95% free of the corresponding(+) enantiomer.

In a further embodiment the compound of the present invention are in theform of the (−) enantiomer at least 97% free of the corresponding (+)enantiomer.

In a further embodiment the compound of the present invention are in theform of the (−) enantiomer at least 99% free of the corresponding (+)enantiomer.

There is also provided pharmaceutically acceptable salts of thecompounds of the present invention. By the term pharmaceuticallyacceptable salts of compounds are meant those derived frompharmaceutically acceptable inorganic and organic acids and bases.Examples of suitable acids include hydrochloric, hydrobromic, sulphuric,nitric, perchloric, fumaric, maleic, phosphoric, glycollic, lactic,salicylic, succinic, toluene-p-sulphonic, tartaric, acetic,trifluoroacetic, citric, methanesulphonic, formic, benzoic, malonic,naphthalene-2-sulphonic and benzenesulphonic acids. Other acids such asoxalic, while not themselves pharmaceutically acceptable, may be usefulas intermediates in obtaining the compounds of the invention and theirpharmaceutically acceptable acid addition salts.

Salts derived from amino acids are also included (e.g. L-arginine,L-Lysine).

Salts derived from appropriate bases include alkali metals (e.g. sodium,lithium, potassium), alkaline earth metals (e.g. calcium, magnesium),ammonium, NR₄+ (where R is C₁₋₄ alkyl) salts, choline and tromethamine.

A reference hereinafter to a compound according to the inventionincludes that compound and its pharmaceutically acceptable salts.

In one embodiment of the invention, the pharmaceutically acceptable saltis a sodium salt.

In one embodiment of the invention, the pharmaceutically acceptable saltis a lithium salt.

In one embodiment of the invention, the pharmaceutically acceptable saltis a potassium salt.

In one embodiment of the invention, the pharmaceutically acceptable saltis a tromethamine salt.

In one embodiment of the invention, the pharmaceutically acceptable saltis an L-arginine salt.

It will be appreciated by those skilled in the art that the compounds inaccordance with the present invention can exist in different polymorphicforms. As known in the art, polymorphism is an ability of a compound tocrystallize as more than one distinct crystalline or “polymorphic”species. A polymorph is a solid crystalline phase of a compound with atleast two different arrangements or polymorphic forms of that compoundmolecule in the solid state. Polymorphic forms of any given compound aredefined by the same chemical formula or composition and are as distinctin chemical structure as crystalline structures of two differentchemical compounds.

It will further be appreciated by those skilled in the art that thecompounds in accordance with the present invention can exist indifferent solvate forms, for example hydrates. Solvates of the compoundsof the invention may also form when solvent molecules are incorporatedinto the crystalline lattice structure of the compound molecule duringthe crystallization process.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

The term “alkyl” represents a linear, branched or cyclic hydrocarbonmoiety. The terms “alkenyl” and “alkynyl” represent a linear, branchedor cyclic hydrocarbon moiety which has one or more double bonds ortriple bonds in the chain. Examples of alkyl, alkenyl, and alkynylgroups include but are not limited to methyl, ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl,tert-pentyl, hexyl, isohexyl, neohexyl, allyl, vinyl, acetylenyl,ethylenyl, propenyl, isopropenyl, butenyl, isobutenyl, hexenyl,butadienyl, pentenyl, pentadienyl, hexenyl, hexadienyl, hexatrienyl,heptenyl, heptadienyl, heptatrienyl, octenyl, octadienyl, octatrienyl,octatetraenyl, propynyl, butynyl, pentynyl, hexynyl, cyclopropyl,cyclobutyl, cyclohexenyl, cyclohexadienyl and cyclohexyl. Whereindicated the “alkyl,” “alkenyl,” and “alkynyl” can be optionallysubstituted such as in the case of haloalkyls in which one or morehydrogen atom is replaced by a halogen, e.g., an alkylhalide. Examplesof haloalkyls include but are not limited to trifluoromethyl,difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl,chloromethyl, trifluoroethyl, difluoroethyl, fluoroethyl,trichloroethyl, dichloroethyl, chloroethyl, chlorofluoromethyl,chlorodifluoromethyl, dichlorofluoroethyl. Aside from halogens, whereindicated, the alkyl, alkenyl or alkynyl groups can also be optionallysubstituted by, for example, oxo, —NR_(d)R_(e), —CONR_(d)R_(e),═NO—R_(e), NR_(d)COR_(e), carboxy, —C(═NR_(d))NR_(e)R_(f), azido, cyano,C₁₋₆ alkyloxy, C₂₋₆ alkenyloxy, C₂₋₆ alkynyloxy,—N(R_(d))C(═NR_(e))—NR_(f)R_(g), hydroxyl, nitro, nitroso,—N(R_(h))CONR_(i)R_(j), S(O)₀₋₂R_(a), C(O)R_(a), C(O)OR_(a),NR_(a)C(O)R_(b), SO₂NR_(a)R_(b), NR_(a)SO₂R_(b), NR_(a)SO₂NR_(b)R_(c),CR_(a)N═OR_(b), and/or NR_(a)COOR_(b), wherein R_(a)-R_(j) are eachindependently H, C₁₋₄ alkyl, C₂₋₄ alkenyl or C₂₋₄ alkynyl.

The terms “cycloalkyl”, and “cycloalkenyl” represent a cyclichydrocarbon alkyl or alkenyl, respectively, and are meant to includemonocyclic (e.g., cyclohexyl), spiro (e.g., spiro[2.3]hexanyl), fused(e.g., bicyclo[4.4.0]decanyl), and bridged (e.g.,bicyclo[2.2.1]heptanyl)hydrocarbon moieties.

The terms “alkoxy,” “alkenyloxy,” and “alkynyloxy” represent an alkyl,alkenyl or alkynyl moiety, respectively, which is covalently bonded tothe adjacent atom through an oxygen atom. Like the alkyl, alkenyl andalkynyl groups, where indicated the alkoxy, alkenyloxy and alkynyloxygroups can also be optionally substituted. Examples include but are notlimited to methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy,sec-butoxy, tert-butoxy, pentyloxy, isopentyloxy, neopentyloxy,tert-pentyloxy, hexyloxy, isohexyloxy, trifluoromethoxy and neohexyloxy.The alkoxy, alkenyloxy, and alkynyloxy groups can be optionallysubstituted by, for example, halogens, oxo, —NR_(d)R_(e),—CONR_(d)R_(e), —NR_(d)COR_(e), carboxy, —C(═NR_(d))NR_(e)R_(f), azido,cyano, —N(R_(d))C(═NR_(e))NR_(f)R_(g), hydroxyl, nitro, nitroso, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, —N(R_(h))CONR_(i)R_(j), S(O)₀₋₂R_(a),C(O)R_(a), C(O)OR_(a), ═NO—R_(e), NR_(a)C(O)R_(b), SO₂NR_(a)R_(b),NR_(a)SO₂R_(b), NR_(a)SO₂NR_(b)R_(c), CR_(a)N═OR_(b), and/orNR_(a)COOR_(b), wherein R_(a)-R_(j) are each independently H, C₁₋₄alkyl, C₂₋₄ alkenyl or C₂₋₄ alkynyl.

The term “aryl” represents a carbocyclic moiety containing at least onebenzenoid-type ring (i.e., may be monocyclic or polycyclic), and whichwhere indicated may be optionally substituted with one or moresubstituents. Examples include but are not limited to phenyl, tolyl,dimethylphenyl, aminophenyl, anilinyl, naphthyl, anthryl, phenanthryl orbiphenyl. The aryl groups can be optionally substituted by, for example,halogens, —NR_(d)R_(e), —CONR_(d)R_(e), —NR_(d)COR_(e), carboxy,—C(═NR_(d))NR_(e)R_(f), azido, cyano, —N(R_(d))C(═NR_(e))NR_(f)R_(g),hydroxyl, nitro, nitroso, —N(R_(h))CONR_(i)R_(j), C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₁₋₆ alkyloxy, C₂₋₆ alkenyloxy, C₂₋₆ alkynyloxy,S(O)₀₋₂R_(a), optionally substituted 5-12 member heteroaryl, optionallysubstituted 6-18 member heteroaralkyl, optionally substituted 3-12member heterocycle, optionally substituted 4-18 memberheterocycle-alkyl, C(O)R_(a), C(O)OR_(a), NR_(a)C(O)R_(b),SO₂NR_(a)R_(b), NR_(a)SO₂R_(b), NR_(a)SO₂NR_(b)R_(c), CR_(a)N═OR_(b),and/or NR_(a)COOR_(b), wherein R_(a)-R_(j) are each independently H,C₁₋₄ alkyl, C₂₋₄ alkenyl or C₂₋₄ alkynyl.

The term “aralkyl” represents an aryl group attached to the adjacentatom by an alkyl, alkenyl or alkynyl. Like the aryl groups, whereindicated the aralkyl groups can also be optionally substituted.Examples include but are not limited to benzyl, benzhydryl, trityl,phenethyl, 3-phenylpropyl, 2-phenylpropyl, 4-phenylbutyl andnaphthylmethyl. Where indicated, the aralkyl groups can be optionallysubstituted by, for example, halogens, —NR_(d)R_(e), —CONR_(d)R_(e),—NR_(d)COR_(e), carboxy, —C(═NR_(d))NR_(e)R_(f), azido, cyano,—N(R_(d))C(═NR_(e))NR_(f)R_(g), hydroxyl, nitro, nitroso,—N(R_(h))CONR_(i)R_(j), C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆alkyloxy, C₂₋₆ alkenyloxy, C₂₋₆ alkynyloxy, S(O)₀₋₂R_(a), optionallysubstituted 5-12 member heteroaryl, optionally substituted 6-18 memberheteroaralkyl, optionally substituted 3-12 member heterocycle,optionally substituted 4-18 member heterocycle-alkyl, C(O)R_(a),C(O)OR_(a), NR_(a)C(O)R_(b), SO₂NR_(a)R_(b), NR_(a)SO₂R_(b),NR_(a)SO₂NR_(b)R_(c), CR_(a)N═OR_(b), and/or NR_(a)COOR_(b), whereinR_(a)-R_(j) are each independently H, C₁₋₄ alkyl, C₂₋₄ alkenyl or C₂₋₄alkynyl.

The term “heterocycle” represents an optionally substituted, nonaromatic, saturated or partially saturated wherein said cyclic moiety isinterrupted by at least one heteroatom selected from oxygen (O), sulfur(S) or nitrogen (N). Heterocycles may be monocyclic or polycyclic rings.Examples include but are not limited to azetidinyl, dioxolanyl,morpholinyl, morpholino, oxetanyl, piperazinyl, piperidyl, piperidino,cyclopentapyrazolyl, cyclopentaoxazinyl, cyclopentafuranyl. Whereindicated, the heterocyclic groups can be optionally substituted by, forexample, halogens, oxo, —NR_(d)R_(e), —CONR_(d)R_(e), ═NO—R_(e),—NR_(d)COR_(e), carboxy, —C(═NR_(d))NR_(e)R_(f), azido, cyano,—N(R_(d))C(═NR_(e))NR_(f)R_(g), hydroxyl, nitro, nitroso,—N(R_(h))CONR_(i)R_(j), C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆-alkynyl, C₇₋₁₂aralkyl, C₆₋₁₂ aryl, C₁₋₆ alkyloxy, C₂₋₆ alkenyloxy, C₂₋₆ alkynyloxy,S(O)₀₋₂R_(a), C₆₋₁₀ aryl, C₆₋₁₀ aryloxy, C₇₋₁₀ arylalkyl, C₆₋₁₀aryl-C₁₋₁₀ alkyloxy, C(O)R_(a), C(O)OR_(a), NR_(a)C(O)R_(b),SO₂NR_(a)R_(b), NR_(a)SO₂R_(b), NR_(a)SO₂NR_(b)R_(c), CR_(a)N═OR_(b),and/or NR_(a)COOR_(b), wherein R_(a)-R_(j) are each independently H,C₁₋₄ alkyl, C₂₋₄ alkenyl or C₂₋₄ alkynyl.

The term “heterocycle-alkyl” represents an optionally substitutedheterocycle group attached to the adjacent atom by an alkyl, alkenyl oralkynyl group. It is understood that in a 5-18 member heterocycle-alkylmoiety, the 5-18 member represent the atoms that are present in both theheterocycle moiety and the alkyl, alkenyl or alkynyl group. For example,the following groups are encompassed by a 7 member heterocycle-alkyl (*represents the attachment point):

Where indicated the heterocycle-alkyl groups can be optionallysubstituted by, for example, halogens, oxo, —NR_(d)R_(e),—CONR_(d)R_(e), —NR_(d)COR_(e), carboxy, —C(═NR_(d))NR_(e)R_(f), azido,cyano, —N(R_(d))C(═NR_(e))NR_(f)R_(g), hydroxyl, nitro, nitroso,—N(R_(h))CONR_(i)R_(j), C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆alkyloxy, C₂₋₆ alkenyloxy, C₂₋₆ alkynyloxy, S(O)₀₋₂R_(a), C₆₋₁₀ aryl,C₆₋₁₀ aryloxy, C₇₋₁₀ arylalkyl, C₆₋₁₀ aryl-C₁₋₁₀ alkyloxy, C(O)R_(a),C(O)OR_(a), NR_(a)C(O)R_(b), ═NO—R_(e), SO₂NR_(a)R_(b), NR_(a)SO₂R_(b),NR_(a)SO₂NR_(b)R_(c), CR_(a)N═OR_(b), and/or NR_(a)COOR_(b), whereinR_(a)-R_(j) are each independently H, C₁₋₄ alkyl, C₂₋₄ alkenyl or C₂₋₄alkynyl.

The term “heteroaryl” represents an optionally substituted aromaticcyclic moiety wherein said cyclic moiety is interrupted by at least oneheteroatom selected from oxygen (O), sulfur (S) or nitrogen (N).Heteroaryls may be monocyclic or polycyclic rings. Examples include butare not limited to azepinyl, aziridinyl, azetyl, diazepinyl,dithiadiazinyl, dioxazepinyl, dithiazolyl, furanyl, isooxazolyl,isothiazolyl, imidazolyl, oxadiazolyl, oxiranyl, oxazinyl, oxazolyl,pyrazinyl, pyridazinyl, pyrimidinyl, pyridyl, pyranyl, pyrazolyl,pyrrolyl, pyrrolidinyl, thiatriazolyl, tetrazolyl, thiadiazolyl,triazolyl, thiazolyl, thienyl, tetrazinyl, thiadiazinyl, triazinyl,thiazinyl, thiopyranyl, furoisoxazolyl, imidazothiazolyl,thienoisothiazolyl, thienothiazolyl, imidazopyrazolyl, pyrrolopyrrolyl,thienothienyl, thiadiazolopyrimidinyl, thiazolothiazinyl,thiazolopyrimidinyl, thiazolopyridinyl, oxazolopyrimidinyl,oxazolopyridyl, benzoxazolyl, benzisothiazolyl, benzothiazolyl,imidazopyrazinyl, purinyl, pyrazolopyrimidinyl, imidazopyridinyl,benzimidazolyl, indazolyl, benzoxathiolyl, benzodioxolyl,benzodithiolyl, indolizinyl, indolinyl, isoindolinyl, furopyrimidinyl,furopyridyl, benzofuranyl, isobenzofuranyl, thienopyrimidinyl,thienopyridyl, benzothienyl, benzoxazinyl, benzothiazinyl, quinazolinyl,naphthyridinyl, quinolinyl, isoquinolinyl, benzopyranyl,pyridopyridazinyl and pyridopyrimidinyl. Where indicated the heteroarylgroups can be optionally substituted by, for example, halogens,—NR_(d)R_(e), —CONR_(d)R_(e), —NR_(d)COR_(e), carboxy,—C(═NR_(d))NR_(e)R_(f), azido, cyano, —N(R_(d))C(═NR_(e))NR_(f)R_(g),hydroxyl, nitro, nitroso, —N(R_(h))CONR_(i)R_(j), C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₁₋₆ alkyloxy, C₂₋₆ alkenyloxy, C₂₋₆ alkynyloxy,S(O)₀₋₂R_(a), C₆₋₁₀ aryl, C₆₋₁₀ aryloxy, C₇₋₁₀ arylalkyl, C₆₋₁₀aryl-C₁₋₁₀ alkyloxy, C(O)R_(a), C(O)OR_(a), NR_(a)C(O)R_(b),SO₂NR_(a)R_(b), NR_(a)SO₂R_(b), NR_(a)SO₂NR_(b)R_(c), CR_(a)N═OR_(b),and/or NR_(a)COOR_(b), wherein R_(a)-R_(j) are each independently H,C₁₋₄ alkyl, C₂₋₄ alkenyl or C₂₋₄ alkynyl.

The term “heteroaralkyl” represents an optionally substituted heteroarylgroup attached to the adjacent atom by an alkyl, alkenyl or alkynylgroup. Where indicated the heteroaralkyl groups can be optionallysubstituted by, for example, halogens, —NR_(d)R_(e), —CONR_(d)R_(e),—NR_(d)COR_(e), carboxy, —C(═NR_(d))NR_(e)R_(f), azido, cyano,—N(R_(d))C(═NR_(e))NR_(f)R_(g), hydroxyl, nitro, nitroso,—N(R_(h))CONR_(i)R_(j), C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆alkyloxy, C₂₋₆ alkenyloxy, C₂₋₆ alkynyloxy, S(O)₀₋₂R_(a), C₆₋₁₀ aryl,C₆₋₁₀ aryloxy, C₇₋₁₀ arylalkyl, C₆₋₁₀ aryl-C₁₋₁₀ alkyloxy, C(O)R_(a),C(O)OR_(a), NR_(a)C(O)R_(b), SO₂NR_(a)R_(b), NR_(a)SO₂R_(b),NR_(a)SO₂NR_(b)R_(c), CR_(a)N═OR_(b), and/or NR_(a)COOR_(b), whereinR_(a)-R_(j) are each independently H, C₁₋₄ alkyl, C₂₋₄ alkenyl or C₂₋₄alkynyl. It is understood that in a 6-18 member heteroaralkyl moiety,the 6-18 member represents the atoms that are present in both theheterocycle moiety and the alkyl, alkenyl or alkynyl groups. Forexample, the following groups are encompassed by a 7 memberheteroaralkyl (* represents the attachment point):

“Halogen atom” is specifically a fluorine atom, chlorine atom, bromineatom or iodine atom.

The term “amidino” represents —C(═NR_(d))NR_(e)R_(f) wherein R_(d),R_(e) and R_(f) are each independently selected from H, C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₆₋₁₂ aryl and C₇₋₁₂ aralkyl, or R_(e) andR_(f) are taken together with the nitrogen to which they are attached toform an optionally substituted 4 to 10 member heterocycle or anoptionally substituted 5-12 member heteroaryl.

The term “guanidino” represents —N(R_(d))C(═NR_(e))NR_(f)R_(g) whereinR_(d), R_(e), R_(f) and R_(g) are each independently selected from H,C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₆₋₁₂ aryl and C₇₋₁₂ aralkyl,or R_(f) and R_(g) are taken together with the nitrogen to which theyare attached to form an optionally substituted 4 to 10 memberheterocycle or an optionally substituted 5-12 member heteroaryl.

The term “amido” represents —CONR_(d)R_(e) and —NR_(d)COR_(e), whereinR_(d) and R_(e) are each independently selected from H, C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₆₋₁₂ aryl and C₇₋₁₂ aralkyl, or R_(d) andR_(e) are taken together with the nitrogen to which they are attached(or the nitrogen atom and CO group in the case of —NR_(d)COR_(e)) toform an optionally substituted 4 to 10 member heterocycle or anoptionally substituted 5-12 member heteroaryl.

The term “amino” represents a derivative of ammonia obtained bysubstituting one or more hydrogen atom and includes —NR_(d)R_(e),wherein R_(d) and R_(e) are each independently selected from H, C₁₋₁₀alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₆₋₁₂ aryl and C₇₋₁₂ aralkyl, orR_(d) and R_(e) are taken together with the nitrogen to which they areattached to form an optionally substituted 4 to 10 member heterocycle oran optionally substituted 5-12 member heteroaryl.

The term “sulfonamido” represents SO₂NR_(d)R_(e), and —NR_(d)SO₂R_(e),wherein R_(d) and R_(e) are each independently selected from H, C₁₋₁₀alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₆₋₁₂ aryl and C₇₋₁₂ aralkyl, orR_(d) and R_(e) are taken together with the nitrogen to which they areattached to form an optionally substituted 4 to 10 member heterocycle oran optionally substituted 5-12 member heteroaryl.

When there is a sulfur atom present, the sulfur atom can be at differentoxidation levels, i.e., S, SO, or SO₂. All such oxidation levels arewithin the scope of the present invention.

The term “independently” means that a substituent can be the same or adifferent definition for each item.

The terms “host” or “patient” means a human, male or female, forexample, a child, an adolescent, or an adult.

It will be appreciated that the amount of a compound of the inventionrequired for use in treatment will vary not only with the particularcompound selected but also with the route of administration, the natureof the condition for which treatment is required and the age andcondition of the patient and will be ultimately at the discretion of theattendant physician or veterinarian. In general however a suitable dosewill be in the range of from about 0.1 to about 750 mg/kg of body weightper day, for example, in the range of 0.5 to 60 mg/kg/day, or, forexample, in the range of 1 to 20 mg/kg/day.

The desired dose may conveniently be presented in a single dose or asdivided dose administered at appropriate intervals, for example as two,three, four or more doses per day.

The compound is conveniently administered in unit dosage form; forexample containing 10 to 1500 mg, conveniently 20 to 1000 mg, mostconveniently 50 to 700 mg of active ingredient per unit dosage form.

Ideally the active ingredient should be administered to achieve peakplasma concentrations of the active compound of from about 1 to about 75μM, about 2 to 50 μM, about 3 to about 30 μM. This may be achieved, forexample, by the intravenous injection of a 0.1 to 5% solution of theactive ingredient, optionally in saline, or orally administered as abolus containing about 1 to about 500 mg of the active ingredient.Desirable blood levels may be maintained by a continuous infusion toprovide about 0.01 to about 5.0 mg/kg/hour or by intermittent infusionscontaining about 0.4 to about 15 mg/kg of the active ingredient.

When the compounds of the present invention or a pharmaceuticallyacceptable salts thereof is used in combination with a secondtherapeutic agent active against the same virus the dose of eachcompound may be either the same as or differ from that when the compoundis used alone. Appropriate doses will be readily appreciated by thoseskilled in the art.

While it is possible that, for use in therapy, a compound of theinvention may be administered as the raw chemical it is preferable topresent the active ingredient as a pharmaceutical composition. Theinvention thus further provides a pharmaceutical composition comprisingcompounds of the present invention or a pharmaceutically acceptablederivative thereof together with one or more pharmaceutically acceptablecarriers therefore and, optionally, other therapeutic and/orprophylactic ingredients. The carrier(s) must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not deleterious to the recipient thereof.

Pharmaceutical compositions include those suitable for oral, rectal,nasal, topical (including buccal and sub-lingual), transdermal, vaginalor parenteral (including intramuscular, sub-cutaneous and intravenous)administration or in a form suitable for administration by inhalation orinsufflation. The formulations may, where appropriate, be convenientlypresented in discrete dosage units and may be prepared by any of themethods well known in the art of pharmacy. All methods include the stepof bringing into association the active compound with liquid carriers orfinely divided solid carriers or both and then, if necessary, shapingthe product into the desired formulation.

Pharmaceutical compositions suitable for oral administration mayconveniently be presented as discrete units such as capsules, cachets ortablets each containing a predetermined amount of the active ingredient;as a powder or granules; as a solution, a suspension or as an emulsion.The active ingredient may also be presented as a bolus, electuary orpaste. Tablets and capsules for oral administration may containconventional excipients such as binding agents, fillers, lubricants,disintegrants, or wetting agents. The tablets may be coated according tomethods well known in the art. Oral liquid preparations may be in theform of, for example, aqueous or oily suspensions, solutions, emulsions,syrups or elixirs, or may be presented as a dry product for constitutionwith water or other suitable vehicle before use. Such liquidpreparations may contain conventional additives such as suspendingagents, emulsifying agents, non-aqueous vehicles (which may includeedible oils), or preservatives.

The compounds according to the invention may also be formulated forparenteral administration (e.g. by injection, for example bolusinjection or continuous infusion) and may be presented in unit dose formin ampoules, pre-filled syringes, small volume infusion or in multi-dosecontainers with an added preservative. The compositions may take suchforms as suspensions, solutions, or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilization from solution, for constitution witha suitable vehicle, e.g. sterile, pyrogen-free water, before use.

For topical administration to the epidermis, the compounds according tothe invention may be formulated as ointments, creams or lotions, or as atransdermal patch. Such transdermal patches may contain penetrationenhancers such as linalool, carvacrol, thymol, citral, menthol andt-anethole. Ointments and creams may, for example, be formulated with anaqueous or oily base with the addition of suitable thickening and/orgelling agents. Lotions may be formulated with an aqueous or oily baseand will in general also contain one or more emulsifying agents,stabilizing agents, dispersing agents, suspending agents, thickeningagents, or colouring agents.

Compositions suitable for topical administration in the mouth includelozenges comprising active ingredient in a flavoured base, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert base such as gelatin and glycerin or sucrose andacacia; and mouthwashes comprising the active ingredient in a suitableliquid carrier.

Pharmaceutical compositions suitable for rectal administration whereinthe carrier is a solid are for example presented as unit dosesuppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art, and the suppositories may beconveniently formed by admixture of the active compound with thesoftened or melted carrier(s) followed by chilling and shaping inmoulds.

Compositions suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or sprays containing inaddition to the active ingredient such carriers as are known in the artto be appropriate.

For intra-nasal administration the compounds of the invention may beused as a liquid spray or dispersible powder or in the form of drops.Drops may be formulated with an aqueous or non-aqueous base alsocomprising one more dispersing agents, solubilizing agents or suspendingagents. Liquid sprays are conveniently delivered from pressurized packs.

For administration by inhalation the compounds according to theinvention are conveniently delivered from an insufflator, nebulizer or apressurized pack or other convenient means of delivering an aerosolspray. Pressurized packs may comprise a suitable propellant such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, thecompounds according to the invention may take the form of a dry powdercomposition, for example a powder mix of the compound and a suitablepowder base such as lactose or starch. The powder composition may bepresented in unit dosage form in, for example, capsules or cartridges ore.g. gelatin or blister packs from which the powder may be administeredwith the aid of an inhalator or insufflator.

When desired the above described formulations adapted to give sustainedrelease of the active ingredient may be employed.

A compound of formula (I) may be prepared by reacting a compound offormula (II):

with a compound of the formula:

under conventional Sonogashira coupling conditions;wherein;

-   X is as defined above, for example, —NR₆—CO—R₅,-   R₁, R₆ and R₅ are as defined herein, Pg₁ is OH or a carboxyl    protecting group, Hal is Cl, Br, or I (e.g., Br),    In a further embodiment, Pg₁ is methoxy or tert-butoxy.    In a further embodiment, Pg₁ is methoxy.

The Sonogashira coupling reaction is a well established method forproducing acetylene containing compounds. Conditions for such couplingare well known in the art and can be found for example in the examplesof the present application in Yamaguchi et al (Synlett 1999, No. 5,549-550) or in Tykwinski et al, Angew. Chem. Int. Ed. 2003, 42,1566-1568.

The present invention also includes intermediates that can be useful inthe synthesis of the compounds of formula (I). Certain intermediates arerepresented by formula W

wherein;

-   R₂, H or amino protecting group (e.g. Boc (tert-butoxycarbonyl), Cbz    (benzyloxycarbonyl)) and Pg₁ is OH or a carboxyl protecting group

In a further embodiment, Pg₁ is methoxy or tert-butoxy.

In a further embodiment, Pg₁ is methoxy.

In a further embodiment, R2 is H.

In a further embodiment, R2 is Boc.

Specific intermediates include but are not limited to compounds listedin Table A:

TABLE A Structure Name #

3-[(1,4-dioxa-spiro[4.5]dec-8- yl)-(trans-4-methyl-cyclo-hexanecarbonyl)-amino]-5-iodo- thiophene-2-carboxylic acid methyl ester3a

5-(3,3-dimethyl-but-1-ynyl)-3- (4-[1,2,3]triazol-1-yl-cyclohexylamino)-thiophene-2- carboxylic acid methyl ester 4a

5-(3,3-dimethyl-but-1-ynyl)-3- (4-[1,2,4]triazol-1-yl-cyclohexylamino)-thiophene-2- carboxylic acid methyl ester 5a

5-(3,3-dimethyl-but-1-ynyl)-3- (tert-butoxycarbonyl)amino-thiophene-2-carboxylic acid methyl ester 9a

5-(3,3-dimethyl-but-1-ynyl)-3- amino-thiophene-2-carboxylic acid methylester 9b

3-[(trans-4-hydroxy- cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-5- iodo-thiophene-2-carboxylic acid methylester 10a

3-[(trans-4-Hydroxy- cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-5- trimethyl-silanylethynyl-thiophene-2-carboxylic acid methyl ester 10b

According to a further aspect of this invention there is providing aprocess for the preparation of compounds of formula I wherein X is

comprising using an intermediate of formula 3a or 10a or 10b.

According to a further aspect of this invention there is providing aprocess for the preparation of compounds of formula I wherein X is

comprising using an intermediate of formula 3a or 10a or 10b.

According to a further aspect of this invention there is providing aprocess for the preparation of compounds of formula I wherein R1 is3,3-dimethyl-but-1-ynyl comprising using an intermediate of formula 9aor 9b.

According to a further aspect of this invention there is providing aprocess for the preparation of compounds of formula I wherein R1 is3,3-dimethyl-but-1-ynyl and R6 is 4-[1,2,3]triazol-1-yl-cyclohexylcomprising using an intermediate of formula 4a.

According to a further aspect of this invention there is providing aprocess for the preparation of compounds of formula I wherein R1 is3,3-dimethyl-but-1-ynyl and R6 is 4-[1,2,4]triazol-1-yl-cyclohexylcomprising using an intermediate of formula 5a.

The following general schemes and examples are provided to illustratevarious embodiments of the present invention and shall not be consideredas limiting in scope. It will be appreciated by those of skill in theart that other compounds of the present invention can be obtained bysubstituting the generically or specifically described reactants and/oroperating conditions used in the following examples. Synthesis methodsto obtain thiophene compounds are also described in patent applicationsU.S. Pat. Nos. 6,881,741, 10/730,272 filed Dec. 9, 2003, U.S. Ser. No.11/042,442 filed Jan. 26, 2005, U.S. Ser. No. 11/433,749 filed May 15,2006, WO02/100851, US 2004-0116509, WO2004/052885, US 2005-0009804,WO2004/052879 and US 2004-0192707. Thiophene alkinyl compounds are alsodisclosed in WO 2006/072347 and WO 2006/072348.

In the foregoing and in the following examples, all temperatures are setforth uncorrected in degrees Celsius; and, unless otherwise indicated,all parts and percentages are by weight.

The following abbreviations may be used as follows:

Boc tert-butoxycarbonyl DCC 1,3-dicyclohexylcarbodiimide DCE1,2-dichloroethane DCM dichloromethane DIPEA N,N-diisopropylethylamineDMF N,N-dimethylformamide EToAc Ethyl acetate Hal halogen LAH lithiumaluminium hydride MeOH Methanol TFA trifluoroacetic acid THFtetrahydrofuran LDA lithium diisopropylamide TLC thin layerchromatography RBO round bottom flask

Purifications by HPLC were all performed using reverse phase C18 columnpacked with 5 m particles. Column diameter was 19 mm and length was 100mm. Eluent was an appropriate gradient of acetonitrile and water with a3 mM HCl concentration.

EXAMPLE 1 Preparation of5-(3,3-Dimethyl-but-1-ynyl)-3-[(trans-4-hydroxy-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]thiophene-2-carboxylicAcid

Step I

A suspension of 3-amino-5-bromo-thiophene-2-carboxylic acid methyl ester(17.0 g, 72.0 mmol) in dry THF (21 mL) is treated with1,4-cyclohexanedione monoethylene ketal (11.3 mg, 72.0 mmol), followedby dibutyltin dichloride (1.098 g, 3.6 mmol). After 5 min, phenyl silane(9.74 mL, 79.2 mmol) is added and the reaction mixture is stirredover-night at room temperature. After concentration, the residue isdissolved in EtOAc washed with NaHCO₃ then brine. The organic layer isseparated, dried on Na₂SO₄, filtered and concentrated. The crudematerial is diluted in hexane (500 mL). After filtration, the motherliquor is evaporated to dryness to give5-bromo-3-(1,4-dioxa-spiro[4.5]dec-8-ylamino)-thiophene-2-carboxylicacid methyl ester (24.79 g, 92% yield).

Ref: WO2004/052885

Step II

A—Preparation of Trans-4-methylcyclohexyl Carboxylic Acid Chloride:

Oxalyl chloride (2M in DCM, 117 mL) is added drop wise to a suspensionof trans-4-methylcyclohexyl carboxylic acid (16.6 g, 117 mmol) in DCM(33 mL) and DMF (0.1 mL), and the reaction mixture is stirred 3 h atroom temperature. DCM is removed under reduced pressure and the residueis co-evaporated with DCM. The residue is dissolved in toluene to make a1M solution.

B—Preparation of the Target Compound:

The 1M solution of trans-4-methylcyclohexyl carboxylic acid chloride isadded to a solution of5-bromo-3-(1,4-dioxa-spiro[4.5]dec-8-ylamino)-thiophene-2-carboxylicacid methyl ester (24.79 g, 65 mmol) in toluene (25 mL) followed bypyridine (5.78 mL, 71.5 mmol). The resulting mixture is then stirred for16 h at reflux. The reaction mixture is diluted with toluene (60 mL) andcooled down to 5° C. After the addition of pyridine (12 mL) and MeOH(5.6 mL), the mixture is stirred 2 h at 5° C. The white suspension isfiltered off and the toluene is added to the mother liquor. The organicphase is washed with 10% citric acid, aq. Sat NaHCO₃, dried (Na₂SO₄) andconcentrated. The residue is triturated in boiling hexane (1500 mL). Thereaction mixture is allowed to cool down to room temperature. Thereaction flask is immersed into ice bath, and stirred for 30 min; whitesolid is filtered off, and washed with cold hexane (225 mL). The solidis purified by silica gel column chromatography using 20% EtOAc:hexaneas eluent to furnish the final compound5-bromo-3-[(1,4-dioxa-spiro[4.5]dec-8-yl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-thiophene-2-carboxylicacid methyl ester (10.5 g, 32%).

Step III

5-Bromo-3-[(1,4-dioxa-spiro[4.5]dec-8-yl)-(trans-4-methylcyclohexane-carbonyl)-amino]-thiophene-2-carboxylicacid methyl ester (8.6 g, 17 mmol) is dissolved in tetrahydrofuran (100mL) and treated with 3N HCl solution (50 mL). The reaction is stirred at40° C. for 3 h. The reaction mixture is evaporated under reducedpressure. The residue is dissolved in EtOAc and washed with aq. sat.NaHCO₃ solution. The organic layer is separated, dried on Na₂SO₄,filtered and concentrated to give5-bromo-3-[(trans-4-methyl-cyclohexanecarbonyl)-(4-oxo-cyclohexyl)-amino]-thiophene-2-carboxylicacid methyl ester as a solid (7.4 g, 95%).

Step IV

To a cold (0° C.) solution of5-bromo-3-[(trans-4-methyl-cyclohexanecarbonyl)-(4-oxo-cyclohexyl)-amino]-thiophene-2-carboxylicacid methyl ester (5.9 g, 12.9 mmol) in 50 mL of MeOH under a N₂atmosphere, NaBH₄ (250 mg, 6.4 mmol) is added portion wise (approx. 30min). After the addition is completed and checked for reactioncompletion by TLC (hexane:EtOAc 1:1), 10 mL of HCl 2% is added andstirred for 15 min. The reaction mixture is concentrated under vacuum todryness. The reaction mixture is recuperated with water (25 mL) andextracted with EtOAC. The organic phases are combined and dried overMgSO₄ and concentrated to dryness. The residue is purified by silica gelcolumn chromatography using EtOAc:hexane (1:1) as eluent to obtain5-bromo-3-[(trans-4-hydroxy-cyclohexyl)-(trans-4-methyl-cyclohexane-carbonyl)-amino]-thiophene-2-carboxylicacid methyl ester (4.5 g, 77% yield) as a solid.

Step V

To a solution of compounds5-bromo-3-[(trans-4-hydroxy-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-thiophene-2-carboxylicacid methyl ester (500 mg, 1.09 mmol) and 3,3-Dimethyl-but-1-yne (385mg, 4.69 mmol) in DMF (0.5 mL), triethylamine (1.06 mL) andtris(dibenzylideneacetone)dipalladium (0) (70 mg, 0.08 mmol) are addedand the reaction mixture is stirred under reflux conditions for 16 hunder a N₂ atmosphere. DMF and triethylamine are removed under reducedpressure and the residue is partitioned between water and ethyl acetate.The organic layer is separated, dried (Na₂SO₄), concentrated and theresidue is purified by column chromatography using ethyl acetate andhexane (1:2) as eluent to obtain5-(3,3-dimethyl-but-1-ynyl)-3-[(trans-4-hydroxy-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-thiophene-2-carboxylicacid methyl ester as a solid, 330 mg (66%).

Step VI

5-(3,3-Dimethyl-but-1-ynyl)-3-[(trans-4-hydroxy-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-thiophene-2-carboxylicacid methyl ester (0.10 g, 0.22 mmol) is dissolved in a 3:2:1 mixture ofTHF:methanol:H₂O (5.0 mL) and treated with a 1N solution of LiOH.H₂O(0.65 mL, 0.65 mmol). After 2 h of stirring at 60° C., the reactionmixture is concentrated under reduced pressure on a rotary evaporator.The mixture is partitioned between ethyl acetate and water. The waterlayer is acidified using 0.1 N HCl. The EtOAc layer is separated anddried over Na₂SO₄. Filtration and removal of the solvent under reducedpressure on a rotary evaporator followed by purification by columnchromatography using methanol and dichloromethane (1:9) as eluent toobtain5-(3,3-dimethyl-but-1-ynyl)-3-[(trans-4-hydroxy-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-thiophene-2-carboxylicacid as a solid, 30 mg (30%). ESI⁻ (M−H): 444.3.

EXAMPLE 2 Preparation of5-(3,3-Dimethyl-but-1-ynyl)-3-[(trans-4-methoxy-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]thiophene-2-carboxylicAcid

Step I:

To a solution of5-(3,3-dimethyl-but-1-ynyl)-3-[(trans-4-hydroxy-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-thiophene-2-carboxylicacid methyl ester (0.200 g, 0.435 mmol) in dry DMF (2.0 mL) is addediodomethane (0.136 mL, 2.18 mmol), the mixture is cooled to 0° C., andNaH (60% suspension in oil, 35 mg, 0.87 mmol) is added in portions over5 min. The mixture is stirred at 0° C. for 1 h 40 min, and it isquenched by addition of water and acidified with 2N HCl. The mixture isdiluted with ethyl acetate and washed with brine. The organic layer isseparated, dried over Na₂SO₄, concentrated under reduced pressure. Theresidue is purified by column chromatography on silica gel eluting with0→50% ethyl acetate in hexane to give5-(3,3-dimethyl-but-1-ynyl)-3-[(trans-4-methoxy-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-thiophene-2-carboxylicacid methyl ester (65 mg, 32%).

Step II:

5-(3,3-Dimethyl-but-1-ynyl)-3-[(trans-4-methoxy-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-thiophene-2-carboxylicacid methyl ester from step I is hydrolysed as previously described(Scheme 1, step VI) to give5-(3,3-dimethyl-but-1-ynyl)-3-[(trans-4-methoxy-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-thiophene-2-carboxylicacid as a solid (65 mg, 32%). ESI⁻ (M−H): 458.3.

EXAMPLE 35-(3,3-Dimethyl-but-1-ynyl)-3-[(trans-4-hydroxy-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-thiophene-2-carboxylicAcid

Step I

A suspension of 3-amino-thiophene-2-carboxylic acid methyl ester (5.0 g,31.85 mmol) in dry THF (9 mL) is treated with 1,4-cyclohexanedionemonoethylene ketal (5.0 g, 32.05 mmol), followed by dibutyltindichloride (482 mg, 1.59 mmol). After 5 min, phenyl silane (4.3 mL,34.96 mmol) is added and the reaction mixture is stirred overnight atroom temperature. After concentration, the residue is dissolved in EtOAcand washed with NaHCO₃ followed by brine. The organic layer isseparated, dried (Na₂SO₄), filtered and concentrated. The residue ispurified by column chromatography using 30% ethyl acetate in hexane aseluent to give3-(1,4-dioxa-spiro[4.5]dec-8-ylamino)-thiophene-2-carboxylic acid methylester (4.5 g, 47% yield).

Alternative Procedure:

3-Amino-thiophene-2-carboxylic acid methyl ester (1 eq.) is dissolved indichloromethane followed by 1,4-cyclohexanedione monoethylene acetal (2eq.) to obtain a slightly yellow solution. This solution is added to thesuspension of NaBH(OAc)₃ (2.2 eq.) in dichloromethane. Acetic acid (2.4eq.) is added dropwise over a period of 15 min. The resulting suspensionis stirred at 20˜25° C. under N₂ for 24 h. The reaction is quenched byadding water and stirred for 1 h. Dichloromethane layer is separated,treated with water again and stirred for another 1 h. Thedichloromethane layer is separated and added to a saturated NaHCO₃solution, stirred at 20˜25° C. for 20 min. Some of the white residualsolids are filtered and then the organic layer is separated, dried(Na₂SO₄) and evaporated. Methanol is added to the residue and evaporatedto dryness. The residue is taken in of methanol and stirred for 2 h at0° C. The suspension is vacuum-filtered and the resulting filtered cakeis washed with cold methanol. The white solid is dried under vacuum at35˜40° C. for approximately 20 h to afford the title compound.

Step II

A—Preparation of trans-4-methylcyclohexyl carboxylic acid chloride:

Oxalyl chloride (2M in dichloromethane, 17 mL) is added dropwise to asuspension of trans-4-methylcyclohexyl carboxylic acid (2.3 g, 16.2mmol) in dichloromethane (5 mL) and DMF (0.1 mL). The reaction mixtureis stirred for 3 h at room temperature. The volatiles are removed underreduced pressure to obtain the crude acid chloride which is useddirectly for the next reaction.

B—trans-4-Methylcyclohexyl carboxylic acid chloride is added to asolution of 3-(1,4-dioxa-spiro[4.5]dec-8-ylamino)-thiophene-2-carboxylicacid methyl ester (2.4 g, 8.08 mmol) in toluene (18 mL) followed bypyridine (0.7 mL). The resulting mixture is then stirred for 16 h atreflux. The reaction mixture is diluted with toluene (7 mL) and cooledto 5° C. After the addition of pyridine (1.5 mL) and MeOH (0.8 mL), themixture is stirred 2 h at 5° C. The white solid is filtered and washedwith toluene. The filtrate is washed with 10% citric acid, aq. NaHCO₃,dried (Na₂SO₄) and concentrated. The solid is purified by silica gelcolumn chromatography using 20% EtOAc:hexane as eluent to obtain3-[(1,4-dioxa-spiro[4.5]dec-8-yl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-thiophene-2-carboxylicacid methyl ester (2.3 g, 68%).

Alternative Procedure:

To a solution of trans-4-methylcyclohexyl carboxylic acid (1.8 eq.) intoluene under nitrogen is added anhydrous DMF. The reaction mixture isstirred and thionyl chloride (2.16 eq.) is added over 3-5 min. Themixture is then stirred for 3 h at rt. When the reaction is completed,toluene is added to the reaction mixture. The solution is thenevaporated under reduced nitrogen pressure to half of its volume. Thesolution is dissolved in toluene to obtain a 1N acid chloride solution.

3-(1,4-Dioxa-spiro[4.5]dec-8-ylamino)-thiophene-2-carboxylic acid methylester (1 eq.) and pyridine (2 eq.) are added to the acid chloride (1N)solution. The reaction mixture is stirred at reflux for 15 h. Once thereaction is completed, the reaction mixture is cooled to roomtemperature, and then methanol and toluene are added to it. The reactionmixture is stirred for 1 h at rt and a saturated aqueous solution ofNaHCO₃ is added. The organic layer is separated, dried (Na₂SO₄) andevaporated to about 4 volumes of solvent. To the solution are added 4volumes of heptane while stirring. The reaction flask is immersed intoan ice bath and stirred for 120 min; a beige solid is filtered off andwashed with cold heptane, then dried over night in the vacuum oven toobtain the title compound.

Step III

n-BuLi (2 eq.) is added dropwise for 10 min to a cold (−40° C.) solutionof diisopropylamine (1 eq.) in dry THF. The reaction mixture is stirredat the same temperature for 30 min. Then a solution of3-[(1,4-dioxa-spiro[4.5]dec-8-yl)-(trans-4-methyl-cyclohexane-carbonyl)-amino]-thiophene-2-carboxylicacid methyl ester (1 eq.) in THF is added dropwise (35 min) keeping theinternal temperature around −40° C. The reaction mixture is stirred for30 min and a solution of iodine (2 eq.) in THF is added dropwise,stirred for 30 min at the same temperature before being added a sat.solution of NH₄Cl. The reaction mixture is diluted with ethyl acetateand water. The organic layer is separated and washed with 5% sodiumthiosulfate solution. The organic layer is separated, dried (Na₂SO₄) andevaporated to a suspension and then added heptane. The suspension isstirred at 0° C. for 30 min, filtered and washed with heptane to obtain3-[(1,4-dioxa-spiro[4.5]dec-8-yl)-(trans-4-methyl-cyclo-hexanecarbonyl)-amino]-5-iodo-thiophene-2-carboxylicacid methyl ester.

MS found (electrospray): (M+H): 548.21

Step IV

To a 25 mL RBF under nitrogen,3-[(1,4-dioxa-spiro[4.5]dec-8-yl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-5-iodo-thiophene-2-carboxylicacid methyl ester (1 eq.), copper iodide (0.025 eq.) andtris(dibenzylideneacetone)dipalladium (0) (0.01 eq.) are taken. DMF,triethylamine (2.5 eq.) and 3,3-dimethyl-but-1-yne (2 eq.) are added andthe reaction mixture is stirred at 40° C. for 2 h under a N₂ atmosphere.The reaction mixture is filtered on celite and washed with ethylacetate. The solution is diluted with water and extracted 2 times withethyl acetate. The organic phases are combined and washed 3 times withwater. The organic layer is separated, dried (Na₂SO₄), evaporated toabout 2 mL and then 8 mL of heptane is added. It is evaporated to 2-4 mLand cooled in an ice bath. The formed white solid is filtered, washedwith heptane and dried in oven to obtain5-(3,3-dimethyl-but-1-ynyl)-3-[(1,4-dioxa-spiro[4.5]dec-8-yl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-thiophene-2-carboxylicacid methyl ester.

Step V

5-(3,3-Dimethyl-but-1-ynyl)-3-[(1,4-dioxa-spiro[4.5]dec-8-yl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-thiophene-2-carboxylicacid methyl ester (1 eq.) is dissolved in tetrahydrofuran and treatedwith 3.6 N HCl solution. The reaction is stirred at 40° C. for 5 h.Water is then added and the reaction mixture is cooled to roomtemperature. The reaction mixture is extracted with ethyl acetate (2×50mL). The combined extracts are washed with 25 mL of aqueous saturatedNaHCO₃ and 2×50 mL of water. The organic layer is concentrated to athick oil and 50 mL of heptane is added to the mixture to precipitatethe desired compound which is filtered to give of5-(3,3-dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexanecarbonyl)-(4-oxo-cyclohexyl)-amino]-thiophene-2-carboxylicacid methyl ester.

Step VI

5-(3,3-Dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexanecarbonyl)-(4-oxo-cyclohexyl)-amino]-thiophene-2-carboxylicacid methyl ester (1 eq.) is dissolved in THF. Water is added to thereaction mixture and cooled to −25° C. NaBH₄ (0.5 eq.) is added portionwise maintaining the temperature below −20° C. The mixture is stirredfor 2 h at −25° C., 2N HCl is then added and the solution is warmed toroom temperature. The phases are separated and the aqueous layer iswashed with EtOAC. The organic phases are combined and washed with brineand dried over Na₂SO₄ and concentrated to dryness to give5-(3,3-dimethyl-but-1-ynyl)-3-[(4-hydroxy-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-thiophene-2-carboxylicacid methyl ester as a 93:7 mixture of isomers. The crude cis/transmixture is recrystallized in methanol to obtain >99% the trans isomer.

Step VII

The same procedure as reported earlier (example 1, step VI) is followedto obtain5-(3,3-dimethyl-but-1-ynyl)-3-[(trans-4-hydroxy-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-thiophene-2-carboxylicacid.

MS found (electrospray): (M−H): 444.3

EXAMPLE 45-(3,3-Dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexanecarbonyl)-(cis-4-[1,2,3]triazol-1-yl-cyclohexyl)-amino]thiophene-2-carboxylicAcid and5-(3,3-Dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexane-carbonyl)-(trans-4-[1,2,3]triazol-1-yl-cyclohexyl)-amino]thiophene-2-carboxylicAcid

Step I:

To a solution of3-amino-5-(3,3-dimethyl-but-1-ynyl)-thiophene-2-carboxylic acid methylester (example 9) (0.387 g, 1.6 mmol) and4-[1,2,3]triazol-1-yl-cyclohexanone (0.27 g, 1.6 mmol) in dry THF isadded dibutyltin dichloride (0.024 g, 0.080 mmol) followed byphenylsilane (0.276 ml, 2.2 mmol). The mixture is stirred overnight atroom temperature. Solvent is evaporated under reduced pressure, and theresidue is diluted with ethyl acetate. The organic layer is washed withwater and brine, dried with sodium sulfate, filtered and concentratedunder reduced pressure. The residue is purified by column chromatographyon silica gel using gradient 50-100% ethyl acetate in hexane to afford5-(3,3-dimethyl-but-1-ynyl)-3-(4-[1,2,3]triazol-1-yl-cyclohexylamino)-thiophene-2-carboxylicacid methyl ester.

Step II:

To a solution of5-(3,3-dimethyl-but-1-ynyl)-3-(4-[1,2,3]triazol-1-yl-cyclohexylamino)-thiophene-2-carboxylicacid methyl ester (0.20 g, 0.50 mmol) in toluene (1 ml) is added asolution of trans-4-methylcyclohexyl carboxylic acid chloride 1 M (1.0ml, 1.0 mmol) and pyridine (0.046 ml, 0.58 mmol). The mixture is stirredovernight at 105° C. and diluted with ethyl acetate. The organic layeris washed with NaHCO_(3 sat) (2×) and brine. The organic layer is driedwith sodium sulfate, filtered and concentrated under reduce pressure.The residue is purified by silica gel column chromatography (20% ethylacetate/hexane to 100% ethyl acetate followed by 10% MeOH/ethyl acetate)to give5-(3,3-dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexanecarbonyl)-(4-[1,2,3]triazol-1-yl-cyclohexyl)-amino]-thiophene-2-carboxylicacid methyl ester.

Step III:

5-(3,3-Dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexanecarbonyl)-(4-[1,2,3]triazol-1-yl-cyclohexyl)-amino]-thiophene-2-carboxylicacid methyl ester (0.13 g, 0.25 mmol) is hydrolyzed with lithiumhydroxide as previously described (example 1, step VI) to give afterHPLC purification the pure isomer5-(3,3-dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexanecarbonyl)-(cis-4-[1,2,3]triazol-1-yl-cyclohexyl)-amino]-thiophene-2-carboxylicacid and5-(3,3-dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexanecarbonyl)-(trans-4-[1,2,3]triazol-1-yl-cyclohexyl)-amino]-thiophene-2-carboxylicacid.

MS found (electrospray): (M+H): 497.4

EXAMPLE 55-(3,3-Dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexanecarbonyl)-(cis-4-[1,2,4]triazol-1-yl-cyclohexyl)-amino]-thiophene-2-carboxylicAcid and5-(3,3-Dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexane-carbonyl)-(trans-4-[1,2,4]triazol-1-yl-cyclohexyl)-amino]-thiophene-2-carboxylicAcid

Step I:

Reductive amination of3-amino-5-(3,3-dimethyl-but-1-ynyl)-thiophene-2-carboxylic acid methylester (0.237 g, 1.0 mmol) and 4-[1,2,4]triazol-1-yl-cyclohexanone (0.170g, 1.0 mmol) is performed under the same conditions previously describedusing dibutyltin dichloride and phenylsilane to give5-(3,3-dimethyl-but-1-ynyl)-3-(4-[1,2,4]triazol-1-yl-cyclohexylamino)-thiophene-2-carboxylicacid methyl ester.

Step II:

5-(3,3-Dimethyl-but-1-ynyl)-3-(4-[1,2,4]triazol-1-yl-cyclohexylamino)-thiophene-2-carboxylicacid methyl ester (0.27 g, 0.70 mmol) is acylated withtrans-4-methylcyclohexyl carboxylic acid chloride as previouslydescribed to give5-(3,3-dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexanecarbonyl)-(4-[1,2,4]triazol-1-yl-cyclohexyl)-amino]-thiophene-2-carboxylicacid methyl ester.

Step III:

5-(3,3-Dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexanecarbonyl)-(4-[1,2,4]triazol-1-yl-cyclohexyl)-amino]-thiophene-2-carboxylicacid methyl ester (0.244 g, 0.48 mmol) is hydrolysed with lithiumhydroxide as previously described (example 1, step VI) to give afterHPLC purification the pure isomer5-(3,3-Dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexanecarbonyl)-(cis-4-[1,2,4]triazol-1-yl-cyclohexyl)-amino]-thiophene-2-carboxylicacid and5-(3,3-Dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexanecarbonyl)-(trans-4-[1,2,4]triazol-1-yl-cyclohexyl)-amino]-thiophene-2-carboxylicacid.

MS found (electrospray): (M+H): 497.4

EXAMPLE 65-(3,3-Dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexanecarbonyl)-(trans-4-[1,2,3]triazol-1-yl-cyclohexyl)-amino]thiophene-2-carboxylicAcid

Step I:

To a solution of5-(3,3-dimethyl-but-1-ynyl)-3-[(cis-4-hydroxy-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-thiophene-2-carboxylicacid methyl ester (0.92 g, 2.0 mmol) in 10 ml of CH₂Cl₂ is added at 0°C. methanesulfonyl chloride (0.31 mL, 4.0 mmol) followed bytriethylamine (0.56 ml, 4.0 mmol). The reaction mixture is stirred atroom temperature for 24 h and treated with water. The aqueous layer isextracted 2 times with CH₂Cl₂. The organic layer is dried with sodiumsulfate, filtered and concentrated under reduced pressure to give5-(3,3-dimethyl-but-1-ynyl)-3-[(cis-4-methanesulfonyloxy-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-thiophene-2-carboxylicacid methyl ester.

Step II:

To a solution of5-(3,3-dimethyl-but-1-ynyl)-3-[(cis-4-methanesulfonyloxy-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-thiophene-2-carboxylicacid methyl ester (1.16 g, 2.00 mmol) in 10 ml of DMF is added sodiumazide (0.65 g, 10 mmol). The reaction mixture is stirred for 48 h at 50°C. The mixture is diluted with ethyl acetate, washed 3 times with waterand 1 time with brine. The organic layer is dried with sodium sulfate,filtered and concentrated under reduce pressure to give3-[(trans-4-azido-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-5-(3,3-dimethyl-but-1-ynyl)-thiophene-2-carboxylicacid methyl ester.

Step III:

A solution of3-[(trans-4-azido-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-5-(3,3-dimethyl-but-1-ynyl)-thiophene-2-carboxylicacid methyl ester (1.0 g, 2.0 mmol) in trimethylsilylacetylene (1.4 ml,10 mmol) is treated in microwave at 120° C. for 2 h. The mixture isconcentrated under reduced pressure and the residue is purified bysilica gel column chromatography (5% ethyl acetate/hexane to 100% ethylacetate) to afford5-(3,3-dimethyl-but-1-ynyl)-3-{(trans-4-methyl-cyclohexanecarbonyl)-[trans-4-(4-trimethylsilanyl-[1,2,3]triazol-1-yl)-cyclohexyl]-amino}-thiophene-2-carboxylicacid methyl ester.

Step IV:

To a solution of5-(3,3-dimethyl-but-1-ynyl)-3-{(trans-4-methyl-cyclohexanecarbonyl)-[trans-4-(4-trimethylsilanyl-[1,2,3]triazol-1-yl)-cyclohexyl]-amino}-thiophene-2-carboxylicacid methyl ester (0.48 g, 0.82 mmol) in THF (2.0 ml) is added TBAF 1.0M in THF (1.23 ml, 1.23 mmol). The reaction mixture is stirred for 24 hand treated with water and saturated ammonium chloride solution. Theaqueous layer is extracted with ethyl acetate. The organic layer iswashed with brine, dried with sodium sulfate, filtered and concentratedunder reduced pressure. The residue is purified by silica gel columnchromatography (50% ethyl acetate/hexane to 100% ethyl acetate) to give5-(3,3-dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexanecarbonyl)-(trans-4-[1,2,3]triazol-1-yl-cyclohexyl)-amino]-thiophene-2-carboxylicacid methyl ester.

Step V:

5-(3,3-Dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexanecarbonyl)-(trans-4-[1,2,3]triazol-1-yl-cyclohexyl)-amino]-thiophene-2-carboxylicacid methyl ester (0.27 g, 0.52 mmol) is hydrolysed with lithiumhydroxide as previously described (example 1, step VI) to give afterHPLC purification5-(3,3-dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexanecarbonyl)-(trans-4-[1,2,3]triazol-1-yl-cyclohexyl)-amino]-thiophene-2-carboxylicacid.

MS found (electrospray): (M+H): 497.4

Intermediate 2: 4-[1,2,3]triazol-1-yl-cyclohexanone

Step I:

A mixture of methanesulfonic acid 1,4-dioxa-spiro[4.5]dec-8-yl ester(2.80 g, 11.9 mmol) and sodium azide (3.86 g, 59.3 mmol) in 50 mL of dryDMF is stirred for 20 h at 100° C. under nitrogen. The final mixture iscooled to room temperature diluted with brine and extracted with threeportions of ether. The organic portions are combined, dried over Na₂SO₄and concentrated to give 8-azido-1,4-dioxa-spiro[4.5]decane.

Step II:

A mixture of 8-azido-1,4-dioxa-spiro[4.5]decane (1.00 g, 5.43 mmol) and1-(trimethylsilyl)propyne (3.76 mL, 27.1 mmol) is submitted to microwaveat 120° C. for 2 h. The mixture is concentrated under vacuum to removethe excess of 1-(trimethylsilyl)propyne and crude1-(1,4-dioxa-spiro[4.5]dec-8-yl)-4-trimethylsilanyl-1H-[1,2,3]triazoleis obtained.

Step III:

A solution of1-(1,4-dioxa-spiro[4.5]dec-8-yl)-4-trimethylsilanyl-1H-[1,2,3]-triazole(1.60 g, 5.68) in 41 mL of dry THF is treated by a 1M solution oftetrabutylammonium fluoride in THF (9.0 mL, 9.0 mmol). The resultingmixture is stirred for 48 h at room temperature under nitrogen. It isdiluted with EtOAc, washed with saturated aqueous ammonium chloride,water and brine, dried over Na₂SO₄ and concentrated to give1-(1,4-dioxa-spiro[4.5]dec-8-yl)-1H-[1,2,3]triazole.

Step IV:

1-(1,4-Dioxa-spiro[4.5]dec-8-yl)-1H-[1,2,3]triazole (1.06 g, 5.06 mmol)is submitted to the same procedure as for intermediate 1 step III toafford 4-[1,2,3]triazol-1-yl-cyclohexanone as a white solid.

Intermediate 1: 4-[1,2,4]triazol-1-yl-cyclohexanone

Step I:

Methanesulfonic acid 1,4-dioxa-spiro[4.5]dec-8-yl ester is preparedaccording to: Cheng, Chen Yu; Wu, Shou Chien; Hsin, Ling Wei; Tam, S.William. Coll. Med., Natl. Taiwan Univ., Taipei, Taiwan. Journal ofMedicinal Chemistry (1992), 35(12), 2243-7.

Step II:

A solution of methanesulfonic acid 1,4-dioxa-spiro[4.5]dec-8-yl ester(567 mg, 2.40 mmol) and 1,2,4-triazole (232 mg, 3.36 mmol) in dry DMF(5.00 mL) is treated with sodium hydride 60% (125 mg, 3.12 mmol) at roomtemperature under nitrogen. The resulting mixture is stirred at 65° C.for 72 h. It is poured in ice water (75 mL), extracted 3 portions of 75mL of EtOAc. The organic portions are combined, dried over anhydrousNa₂SO₄ and concentrated. The solid is purified by silica gel columnchromatography using a gradient from 100% EtOAc to 5% MeOH:EtOAc aseluent to furnish the final compound1-(1,4-dioxa-spiro[4.5]dec-8-yl)-1H-[1,2,4]triazole as a white solid.

Step III:

1-(1,4-Dioxa-spiro[4.5]dec-8-yl)-1H-[1,2,4]triazole (379 mg, 1.81 mmol)is dissolved in a 1:1 mixture of THF and 3N HCl aqueous solution (9 mL).The resulting mixture is stirred at 40° C. for 5 h. Most of the THF isremoved under vacuum then the remaining mixture is neutralized using a3N NaOH aqueous solution until a basic pH is reached. It is extractedwith 3 portions of 10 mL of dichloromethane. The organic portions arecombined, dried over anhydrous Na₂SO₄ and concentrated to afford4-[1,2,4]triazol-1-yl-cyclohexanone as a white waxy solid.

EXAMPLE 7 Preparation of5-(3,3-dimethyl-but-1-ynyl)-3-[(trans-4-fluoro-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-thiophene-2-carboxylicAcid

To a suspension of5-(3,3-dimethyl-but-1-ynyl)-3-[(cis-4-hydroxy-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-thiophene-2-carboxylicacid (102 mg, 0.23 mmol) in dry CH₂Cl₂ (2 mL) is added DAST(Diethylaminosulphurtrifluoride) (90 μL, 0.69 mmol), and the mixture isstirred for 4 h at room temperature. Then it is diluted with CH₂Cl₂,water is added to the mixture, and it is vigorously stirred for 20 min.Organic fraction is separated, dried over Na₂SO₄, concentrated, and theresidue is purified by preparative HPLC to give5-(3,3-dimethyl-but-1-ynyl)-3-[(trans-4-fluoro-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-thiophene-2-carboxylicacid.

MS found (electrospray): [M+H]: 448.30

EXAMPLE 8 Preparation of5-(3,3-dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexanecarbonyl)-(1-methyl-piperidin-4-yl)-amino]-thiophene-2-carboxylicAcid Hydrochloride

Step I:

To a solution of3-(tert-butoxycarbonyl)amino-5-bromo-thiophene-2-carboxylic acid methylester (4.566 g, 13.58 mmol) in dry DMF (40 mL) are added copper (I)iodide (52 mg, 0.27 mmol), Pd₂ dba₃ (622 mg, 0.68 mmol) andtriethylamine (9.46 mL, 67.9 mmol), and the mixture is deoxygenated bybubbling nitrogen through solution for 10 min. Then tert-butylacetylene(6.62 mL, 54.32 mmol) and BINAP (676 mg, 1.09 mmol) are added to themixture, and it is heated at 60° C. overnight under nitrogen. Themixture is diluted with CH₂Cl₂ and filtered through celite washing withCH₂Cl₂. Filtrate is washed with brine, organic fraction is separated,dried over Na₂SO₄, concentrated, and the residue is purified by columnchromatography on silica gel eluting with gradient of EtOAc in hexane togive of5-(3,3-dimethyl-but-1-ynyl)-3-(tert-butoxycarbonyl)amino-thiophene-2-carboxylicacid methyl ester.

¹H NMR (CDCl₃), ppm: 1.27 (s, 9H), 1.51 (s, 9H), 3.84 (s, 3H), 7.87 (s,1H), 9.24 (br.s, 1H)

MS found (electrospray): [M+H] 338.17

Step II:

To a solution of5-(3,3-dimethyl-but-1-ynyl)-3-(tert-butoxycarbonyl)amino-thiophene-2-carboxylicacid methyl ester (4.344 g, 9.58 mmol) in CH₂Cl₂ (30 mL) is addedtrifluoroacetic acid (30 mL), and the mixture is stirred at roomtemperature overnight. Then it is evaporated to dryness, obtainedresidue is redissolved in CH₂Cl₂ and washed with aqueous NaHCO₃ andbrine. Organic fraction is separated, dried over Na₂SO₄, andconcentrated to give 3.135 g of crude5-(3,3-dimethyl-but-1-ynyl)-3-amino-thiophene-2-carboxylic acid methylester.

¹H NMR (CDCl₃), ppm: 1.28 (s, 9H), 3.80 (s, 3H), 5.36 (br.s, 2H), 6.49(s, 1H)

MS found (electrospray): [M+H] 238.11

Step III:

To a solution of5-(3,3-dimethyl-but-1-ynyl)-3-amino-thiophene-2-carboxylic acid methylester (1.512 g, 5.97 mmol) and N-tert-butoxycarbonyl-piperidin-4-one(1.189 g, 5.97 mmol) in 2 mL of dry THF is added dibutyltin dichloride(181 mg, 0.60 mmol), and the mixture is stirred for 10 min at roomtemperature under nitrogen. Then phenylsilane (810 μL, 6.57 mmol) isadded, and the mixture is stirred for 24 h at room temperature.Additional 595 mg of N-tert-butoxycarbonyl-piperidin-4-one, 90 mg ofdibutyltin dichloride and 405 μL of phenylsilane are added, and themixture is stirred for another 24 h. Then the mixture is diluted withCH₂Cl₂, washed with brine, organic fraction is separated, dried overNa₂SO₄, and concentrated to give 5.142 g of crude5-(3,3-dimethyl-but-1-ynyl)-3-(N-tert-butoxycarbonyl-piperidin-4-yl)amino-thiophene-2-carboxylicacid methyl ester.

Step IV:

trans-4-Methylcyclohexyl carboxylic acid chloride (23.88 mmol) andpyridine (2.89 mL, 35.82 mmol) are added to a solution of5-(3,3-dimethyl-but-1-ynyl)-3-(N-tert-butoxycarbonyl-piperidin-4-yl)amino-thiophene-2-carboxylicacid methyl ester from step III (5.142 g) in dry toluene (50 mL). Themixture is refluxed for 24 h, then it is brought to room temperature,and additional amount of pyridine (1.0 mL) and MeOH (5 mL) are added.Then the mixture is diluted with CH₂Cl₂, washed with brine, organicfraction is separated, dried over Na₂SO₄, and concentrated to give 5.198g of crude5-(3,3-dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexanecarbonyl)-(N-tert-butoxycarbonyl-piperidin-4-yl)amino]-thiophene-2-carboxylicacid methyl ester containing variable amounts of5-(3,3-dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexanecarbonyl)-(piperidin-4-yl)-amino]-thiophene-2-carboxylicacid methyl ester.

Step V:

The product from step IV (5.198 g) is dissolved in 30 mL of CH₂Cl₂ andtreated with 20 mL of trifluoroacetic acid. The mixture is stirred atroom temperature overnight, then it is evaporated to dryness, obtainedresidue is redissolved in CH₂Cl₂ and washed with aqueous NaHCO₃ andbrine. Organic fraction is separated, dried over Na₂SO₄, andconcentrated to give 5.340 g of crude5-(3,3-dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexanecarbonyl)-(piperidin-4-yl)amino]-thiophene-2-carboxylicacid methyl ester.

Step VI:

To a solution of the product from step V (5.340 g) in 1,2-dichloroethane(60 mL) is added formaldehyde (1.94 mL of 37% aqueous solution, 23.88mmol), followed by sodium triacetoxyborohydride (2.403 g, 11.34 mmol) inportions over 20 min. The mixture is stirred at room temperatureovernight, then water is added to the mixture, and it is extracted withCH₂Cl₂. Organic fraction is washed with aqueous NaHCO₃ and brine, driedover Na₂SO₄, concentrated and purified by column chromatography onsilica gel eluting with 0-10% of MeOH in CH₂Cl₂ to give5-(3,3-dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexanecarbonyl)-(1-methyl-piperidin-4-yl)-amino]-thiophene-2-carboxylicacid methyl ester.

Step VII:

The product from Step VI (281 mg, 0.61 mmol) is hydrolysed with lithiumhydroxide as previously described (example 1, step VI) to give afterHPLC purification5-(3,3-dimethyl-but-1-ynyl)-3-[(trans-4-methyl-cyclohexanecarbonyl)-(1-methyl-piperidin-4-yl)-amino]-thiophene-2-carboxylicacid hydrochloride.

MS found (electrospray): [M+H]: 445.29

EXAMPLE 95-(3,3-Dimethyl-but-1-ynyl)-3-[(trans-4-hydroxy-cyclohexyl)-(trans-4-methyl-cyclohexane-carbonyl)-amino]thiophene-2-carboxylicAcid

Step I

3-[(1,4-Dioxa-spiro[4.5]dec-8-yl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-5-iodo-thiophene-2-carboxylicacid methyl ester (from example 3) is dissolved in tetrahydrofuran andtreated with 3N HCl solution. The reaction is stirred at 40° C. for 3 h.The reaction mixture is evaporated under reduced pressure. The mixtureis dissolved in EtOAc and washed with aq. sat. NaHCO₃ solution. Theorganic layer is separated, dried over Na₂SO₄, filtered and concentratedto obtain the title compound.

Step II

To a cold (0° C.) solution of5-iodo-3-[(trans-4-methyl-cyclohexanecarbonyl)-(4-oxo-cyclohexyl)-amino]-thiophene-2-carboxylicacid methyl ester in MeOH under a N₂ atmosphere, NaBH₄ is added portionwise and is stirred. After the reaction is completed, 2% HCl is addedand stirred for 15 min. The reaction mixture is concentrated undervacuum to dryness. The residue is partitioned between water and EtOAC.The organic layer is separated, dried over MgSO₄ and concentrated todryness. The residue is purified by silica gel column chromatographyusing EtOAc:hexane as eluent to obtain the title compound.

Step III

To a solution of3-[(trans-4-hydroxy-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-5-iodo-thiophene-2-carboxylicacid methyl ester and ethynyl-trimethyl-silane in DMF, triethylamine andtris(dibenzylideneacetone)dipalladium (0) are added and the reactionmixture is stirred at 60° C. for 16 h under a N₂ atmosphere. DMF andtriethylamine are removed under reduced pressure and the residue ispartitioned between water and ethyl acetate. The organic layer isseparated, dried (Na₂SO₄), concentrated and the residue is purified bycolumn chromatography using ethyl acetate and hexane (1:2) as eluent toobtain the title compound.

Step IV

3-[(trans-4-Hydroxy-cyclohexyl)-(trans-4-methyl-cyclohexanecarbonyl)-amino]-5-trimethyl-silanylethynyl-thiophene-2-carboxylicacid methyl ester and 2-chloro-2-methylpropane are taken indichloromethane and added freshly sublimed aluminium chloride at −78° C.The reaction mixture is stirred at the same temperature for 6 h. Thereaction mixture is poured into water, diluted with dichloromethane. Theorganic layer is separated, dried (Na₂SO₄) and concentrated. The residueis purified by column chromatography using ethyl acetate and hexane toobtain the title compound.

Ref.: J. Chem. Soc., Chem. Commun., 1982, 959-960.

Step V

5-(3,3-Dimethyl-but-1-ynyl)-3-[(trans-4-hydroxy-cyclohexyl)-(trans-4-methyl-cyclohexane-carbonyl)-amino]-thiophene-2-carboxylicacid methyl ester is dissolved in a 3:2:1 mixture of THF:methanol:H₂Oand treated with a 1N solution of LiOH.H₂O. After 2 h of stirring at 60°C., the reaction mixture is concentrated under reduced pressure on arotary evaporator. The mixture is partitioned between ethyl acetate andwater. The water layer is acidified using 0.1 N HCl. The EtOAc layer isseparated and dried over Na₂SO₄. The solvent is removed and the residueis purified by column chromatography using methanol and dichloromethane(1:9) as eluent to obtain the title compound.

TABLE 1 List of compounds in accordance with the present inventionStructure Chemical Name Mass* 1

5-(3,3-DIMETHYL-BUT-1- YNYL)-3-[(TRANS-4- HYDROXY-CYCLOHEXYL)-(TRANS-4-METHYL- CYCLOHEXANECARBONYL)- AMINO]-THIOPHENE-2- CARBOXYLICACID (M − H): 444.3 2

5-(3,3-DIMETHYL-BUT-1- YNYL)-3-[(TRANS-4- METHOXY-CYCLOHEXYL)-(TRANS-4-METHYL- CYCLOHEXANECARBONYL)- AMINO]-THIOPHENE-2- CARBOXYLICACID (M − H): 458.3. 3

5-(3,3-DIMETHYL-BUT-1- YNYL)-3-[(TRANS-4-METHYL- CYCLOHEXANECARBONYL)-(CIS-4-[1,2,4]TRIAZOL-1-YL- CYCLOHEXYL)-AMINO]- THIOPHENE-2-CARBOXYLICACID (M + H): 497.4 4

5-(3,3-DIMETHYL-BUT-1- YNYL)-3-[(TRANS-4-METHYL- CYCLOHEXANECARBONYL)-(TRANS-4-[1,2,4]TRIAZOL-1- YL-CYCLOHEXYL)-AMINO]- THIOPHENE-2-CARBOXYLICACID (M + H): 497.4 5

5-(3,3-DIMETHYL-BUT-1- YNYL)-3-[(CIS-4-HYDROXY- CYCLOHEXYL)-(TRANS-4-METHYL- CYCLOHEXANECARBONYL)- AMINO]-THIOPHENE-2- CARBOXYLIC ACID (M −H): 444.52 6

5-(3,3-DIMETHYL-BUT-1- YNYL)-3-[(TRANS-4-METHYL- CYCLOHEXANECARBONYL)-(1-METHYL-PIPERIDIN-4-YL)- AMINO]-THIOPHENE-2- CARBOXYLIC ACID;HYDROCHLORIDE (M + H): 445.29 7

5-(3,3-DIMETHYL-BUT-1- YNYL)-3-[(TRANS-4-METHYL- CYCLOHEXANECARBONYL)-(4-CIS-[1,2,3]TRIAZOL-1-YL- CYCLOHEXYL)-AMINO]- THIOPHENE-2-CARBOXYLICACID (M + H): 497.4 8

5-(3,3-DIMETHYL-BUT-1- YNYL)-3-[(TRANS-4-METHYL- CYCLOHEXANECARBONYL)-(TRANS-4-[1,2,3]TRIAZOL-1- YL-CYCLOHEXYL)-AMINO]- THIOPHENE-2-CARBOXYLICACID (M + H): 497.4 9

5-(3,3-DIMETHYL-BUT-1- YNYL)-3-[(TRANS-4-FLUORO- CYCLOHEXYL)-(TRANS-4-METHYL- CYCLOHEXANECARBONYL)- AMINO]-THIOPHENE-2- CARBOXYLIC ACID (M +H): 448.30 *mass spectral analyses are recorded using electrospray massspectrometry.

EXAMPLE 10 Evaluation of Compounds in the HCV RNA-Dependent RNAPolymerase Assay

The following references are all incorporated by reference:

-   -   1. Behrens, S., Tomei, L., De Francesco, R. (1996) EMBO 15,        12-22    -   2. Harlow, E, and Lane, D. (1988) Antibodies: A Laboratory        Manual. Cold Spring Harbord Laboratory. Cold Spring Harbord.        N.Y.    -   3. Lohmann, V., Körner, F., Herian, U., and        Bartenschlager, R. (1997) J. Virol. 71, 8416-8428    -   4. Tomei, L., Failla, C., Santolini, E., De Francesco, R., and        La Monica, N. (1993) J Virol 67, 4017-4026

Compounds are evaluated using an in vitro polymerase assay containingpurified recombinant HCV RNA-dependent RNA polymerase (NS5B protein).HCV NS5B is expressed in insect cells using a recombinant baculovirus asvector. The experimental procedures used for the cloning, expression andpurification of the HCV NS5B protein are described below. Follows, aredetails of the RNA-dependent RNA polymerase assays for testing thecompounds.

Expression of the HCV NS5B Protein in Insect Cells:

The cDNA encoding the entire NS5B protein of HCV-Bk strain, genotype 1b,was amplified by PCR using the primers NS5Nhe5′(5′-GCTAGCGCTAGCTCAATGTCCTACACATGG-3′) and XhoNS53′(5′-CTCGAGCTCGAGCGTCCATCGGTTGGGGAG-3′) and the plasmid pCD 3.8-9.4 astemplate (Tomei et al, 1993). NS5Nhe5′ and XhoNS53′ contain two NheI andXhoI sites (underlined sequences), respectively, at their 5′ end. Theamplified DNA fragment was cloned in the bacterial expression plasmidpET-21b (Novagen) between the restriction sites NheI and XhoI, togenerate the plasmid pET/NS5B. This plasmid was later used as templateto PCR-amplify the NS5B coding region, using the primers NS5B-H9(5′-ATACATATGGCTAGCATGTCAATGTCCTACACATGG-3′) and NS5B-R4(5′-GGATCCGGATCCCGTTCATCGGTTGGGGAG-3′). NS5B-H9 spans a region of 15nucleotides in the plasmid pET-21b followed by the translationinitiation codon (ATG) and 8 nucleotides corresponding to the 5′ end ofthe NS5B coding region (nt. 7590-7607 in the HCV sequence with theaccession number M58335). NS5B-R4 contains two BamHI sites (underlined)followed by 18 nucleotides corresponding to the region around the stopcodon in the HCV genome (nt. 9365-9347). The amplified sequence, of 1.8kb, was digested with NheI and BamHI and ligated to a predigestedpBlueBacII plasmid (Invitrogen). The resulting recombinant plasmid wasdesignated pBac/NS5B. Sf9 cells were co-transfected with 3 μg ofpBac/NS5B, together with 1 μg of linearized baculovirus DNA(Invitrogen), as described in the manufacturer's protocol. Following tworounds of plaque purification, an NS5B-recombinant baculovirus, BacNS5B,was isolated. The presence of the recombinant NS5B protein wasdetermined by western blot analysis (Harlow and Lane, 1988) ofBacNS5B-infected Sf9 cells, using a rabbit polyclonal antiserum(anti-NS5B) raised against a His-tagged version of the NS5B proteinexpressed in E. coli. Infections of Sf9 cells with this plaque purifiedvirus were performed in one-liter spinner flasks at a cell density of1.2×10⁶ cells/ml and a multiplicity of infection of 5.

Preparation of a Soluble Recombinant NS5B Protein:

Sf9 cells were infected as described above. Sixty hours post-infection,cells were harvested then washed twice with phosphate buffer saline(PBS). Total proteins were solubilized as described in Lohmann et al.(1997) with some modifications. In brief, proteins were extracted inthree steps, S1, S2, S3, using lysis buffers (LB) I, LB II and LB III(Lohmann et al, 1997). The composition of LBII was modified to contain0.1% triton X-100 and 150 mM NaCl to reduce the amount of solubilizedNS5B protein at this step. In addition, sonication of cell extracts wasavoided throughout the protocol to preserve the integrity of the proteinstructure.

Purification of Recombinant NS5B Using Fast Protein LiquidChromatography (FPLC):

Soluble NS5B protein in the S3 fraction was diluted to lower the NaClconcentration to 300 mM, then it incubated batchwise with DEAE sepharosebeads (Amersham-Pharmacia) for 2 hrs at 4° C., as described by Behrenset al. (1996). Unbound material was cleared by centrifugation for 15 minat 4° C., at 25 000 rpm using a SW41 rotor (Beckman). The supernatantwas further diluted to lower the NaCl concentration to 200 mM andsubsequently loaded, with a flow rate of 1 ml/min, on a 5 ml HiTrap®heparin column (Amersham-Pharmacia) connected to an FPLC® system(Amersham-Pharmacia). Bound proteins were eluted in 1 ml fractions,using a continuous NaCl gradient of 0.2 to 1 M, over a 25 ml volume.NS5B-containing fractions were identified by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), followed by westernblotting using the anti-NS5B antiserum at a dilution of 1:2000. Positivefractions were pooled and the elution buffer was exchanged against a 50mM NaPO₄ pH 7.0, 20% glycerol, 0.5% triton X-100 and 10 mM DTT, using aPD-10 column (Amersham-Pharmacia). The sample was then loaded onto a 1ml HiTrap® SP column (Amersham-Pharmacia), with a flow rate of 0.1ml/min. Bound proteins were eluted using a continuous 0 to 1 M NaClgradient over a 15 ml volume. Eluted fractions were analyzed by SDS-PAGEand western blotting. Alternatively, proteins were visualized, followingSDS-PAGE, by silver staining using the Silver Stain Plus kit (BioRad) asdescribed by the manufacturer. Positive fractions were tested for RdRpactivity (see below) and the most active ones were pooled, and stored asa 40% glycerol solution at −70° C.

In Vitro HCV RdRp Flashplate Scintillation Proximity Assay (Strep-FlashAssay) Used to Evaluate Analogues:

This assay consists on measuring the incorporation of [³H] radiolabelledUTP in a polyrA/biotinylated-oligo dT template-primer, captured on thesurface of streptavidin-coated scintillant-embeded microtiterFlashplates™ (NEN Life Science Products inc, MA, USA, SMP 103A). Inbrief, a 400 ng/μl polyrA solution (Amersham Pharmacia Biotech) wasmixed volume-to-volume with 5′ biotin-oligo dT₁₅ at 20 pmol/μl. Thetemplate and primers were denatured at 95 C for 5 minutes then incubatedat 37 C for 10 minutes. Annealed template-primers were subsequentlydiluted in a Tris-HCl containing buffer and allowed to bind tostreptavidin-coated flashplates overnight. Unbound material wasdiscarded; compounds were added in a 10 μl solution followed by a 10 μlof a solution containing 50 mM MgCl₂, 100 mM Tris-HCl pH 7.5, 250 mMNaCl and 5 mM DTT. The enzymatic reaction was initiated upon addition ofa 30 μl solution containing the enzyme and substrate to obtain thefollowing concentrations: 25 μM UTP, 1 μCi [³H] UTP and 100 nMrecombinant HCV NS5B. RdRp reactions were allowed to proceed for 2 hrsat room temperature after which wells were washed three times with a 250μL of 0.15 M NaCl solution, air dried at 37 C, and counted using aliquid scintillation counter (Wallac Microbeta Trilex, Perkin-Elmer, MA,USA).

EXAMPLE 11 Cell-Based Luciferase Reporter HCV RNA Replication Assay CellCulture

The compounds of the present invention are HCV polymerase inhibitors.Surprisingly, it has been found that the compounds according to thepresent invention and having a specific substitution pattern, exhibit animproved therapeutic index relative to other thiophene analogues.

Replicon cell lines Huh-7, 5.2 and ET which are derived from the Huh-7hepatocarcinoma cell line were maintained in culture as generallydescribed in Krieger, N; Lohmann, V; Bartenschlager, R. Enhancement ofhepatitis C virus RNA replication by cell culture-adaptive mutations. J.Virol. 2001, 75, 4614-4624. The Huh-7, 5.2 cells contain the highly cellculture-adapted replicon I₃₈₉luc-ubi-neo/NS3-3′/5.1 construct thatcarries, in addition to the neomycin gene, an integrated copy to thefirefly luciferase gene (Krieger, N; Lohmann, V; Bartenschlager, R.Enhancement of hepatitis C virus RNA replication by cellculture-adaptive mutations. J. Virol. 2001, 75, 4614-4624). This cellline allows measurement of HCV RNA replication and translation bymeasuring luciferase activity. It has been previously shown that theluciferase activity tightly follows the replicon RNA level in thesecells (Krieger, N; Lohmann, V; Bartenschlager, R. Enhancement ofhepatitis C virus RNA replication by cell culture-adaptive mutations. J.Virol. 2001, 75, 4614-4624). The Huh-7, ET cell line has the samefeatures as those mentioned for Huh-7, 5.2 cell line, except that ETcells are more robust and contain an adaptative mutation in the HCV NS4Bgene instead of NS5A. Both cell lines were maintained in cultures at asub-confluent level (<85%) as the level of replicon RNA is highest inactively proliferating cells. The culture media used for cell passagingconsist of DMEM (Gibco BRL Laboratories, Mississauga, ON, Canada)supplemented with 10% foetal bovine serum with 1%penicillin/streptomycin, 1% glutamine, 1% sodium pyruvate, 1%non-essential amino acids, and 350 ug/ml of G418 final concentration.Cells were incubated at 37° C., in an atmosphere of 5% CO₂ and passagedtwice a week to maintain sub-confluence.

Approximately 3000 viable Huh-7, 5.2 or ET cells (100 μl) were platedper well in a white opaque 96-well microtiter plate. The cell culturemedia used for the assay was the same as described above except that itcontains no G418 and no phenol red. After an incubation period of 3-4hours at 37° C. in a 5% CO₂ incubator, compounds (100 μl) were added atvarious concentrations. Cells were then further incubated for 4 days at37° C. in a 5% CO₂ incubator. Thereafter, the culture media was removedand cells were lysed by the addition of 95 μL of the luciferase buffer(luciferin substrate in buffered detergent). Cell lysates were incubatedat room temperature and protected from direct light for at least 10minutes. Plates were read for luciferase counts using a luminometer(Wallac MicroBeta Trilux, Perkin Elmer™, MA, USA).

The 50% inhibitory concentrations (IC₅₀s) for inhibitory effect weredetermined from dose response curves using eleven concentrations percompound in duplicate. Curves were fitted to data points using nonlinearregression analysis, and IC₅₀s were interpolated from the resultingcurve using GraphPad Prism software, version 2.0 (GraphPad SoftwareInc., San Diego, Calif., USA).

EXAMPLE 12 Evaluation of Compounds in 21 Amino Acid C-Terminal TruncatedHCV NS5B Genotype 1b Strain BK Enzyme ASSAY

The following references are all incorporated by reference:

-   -   Tomei, L., Failla, C., Santolini, E., De Francesco, R., and La        Monica, N. (1993) J Virol 67, 4017-4026    -   Lesburg, C. A. et al. Crystal structure of the RNA-dependent RNA        polymerase from hepatitis C virus reveals a fully encircled        active site. Nat. Struct. Biol. 6, 937-943 (1999).    -   Ferrari, E. et al. Characterization of soluble hepatitis C virus        RNA-dependent RNA polymerase expressed in Escherichia coli. J.        Virol. 73, 1649-1654 (1999).

Compounds are evaluated using an in vitro polymerase assay containingpurified recombinant HCV RNA-dependent RNA polymerase (NS5B protein)expressed in bacterial cells. The experimental procedures used for thecloning, expression and purification of the HCV NS5B protein aredescribed below. Follows, are details of the RNA-dependent RNApolymerase assays for testing the compounds.

Expression of the HCV NS5B Protein in Insect Cells:

Expression and Purification of HCV NS5B Protein

A recombinant soluble form representing a 21 amino acid C-terminaltruncated HCV NS5B genotype 1b strain BK enzyme (Tomei et al, 1993)containing an N-terminal hexahistidine tag was cloned and expressed inEscherichia coli BL21 (DE3). The truncated enzyme was purified asdescribed in Lesburg et al. (1999) and Ferrari et al. (1999) with minormodifications. Briefly, soluble bacterial lysates were loaded onto aHiTrap nickel chelating affinity column (GE Healthcare, Baie d'Urfe, QC,Canada). The bound enzyme was eluted using an imidazole gradient.Imidazole was then removed from the buffer of the pooled activefractions using PD-10 desalting columns (GE Healthcare, Baie d'Urfe, QC,Canada). Further purification was achieved by running the proteinpreparation through a cation exchange HiTrap SP sepharose column (GEHealthcare, Baie d'Urfe, QC, Canada) using a NaCl gradient for elution.Thereafter, buffer was changed to 10 mM Tris pH 7.5, 10% glycerol, 5 mMDTT, 600 mM NaCl using a PD-10 column. Positive fractions were testedfor RNA-dependent polymerase activity and the most active fractions werepooled and stored at −70° C.

In Vitro NS5B Assay

Measurement of the inhibitory effect of compounds on HCV NS5Bpolymerization activity was performed by evaluating the amount ofradiolabeled UTP incorporated by the enzyme in a newly synthesized RNAusing a homopolymeric RNA template/primer. Briefly, a 15-mer 5′biotinylated DNA oligonucleotide (oligo dT) primer annealed to ahomopolymeric poly rA RNA template is captured on the surface ofstreptavidin-coated bead (GE Healthcare, Baie d'Urfe, QC, Canada).Essentially, compounds were tested at a variety of concentrations (0.005to 200 μM) in a final volume of 50 μL reaction mixture consisting of 20mM Tris-HCl pH 7.5, 5 mM MgCl2, 1 mM DTT, 50 mM NaCl, 50 nM purifiedNS5B enzyme, 250 ng of polyrA/oligodT15 (Invitrogen, Burlington,Ontario, Canada), 15-μM of nonradioactive UTP, and 1 μCi of [³H]UTP(3000 Ci/mmol; GE Healthcare, Baie d'Urfe, QC, Canada). Thepolymerization activity of the HCV NS5B enzyme is quantified bymeasuring the incorporation of radiolabeled [3H]UTP substrate onto thegrowing primer 3′ end and detection is achieved by counting the signalusing a liquid scintillation counter (Wallac MicroBeta Trilux, PerkinElmer™, MA, USA).

[3H]Thymidine Incorporation Assay

A total of 1,000-2,000 cells/well are seeded in 96-well cluster dishesin a volume of 150 μl of DMEM (Life Technologies, Inc., Gaithersburg,Md.) supplemented with 10% FBS (HyClone Laboratories, Inc., Logan, Utah)and 2 mM glutamine (Life Technologies, Inc.). Penicillin andstreptomycin (Life Technologies, Inc.) are added to 500 U/mL and 50μg/mL final concentrations, respectively. After an incubation of 18 h at37° C. in an atmosphere of 5% CO2, the medium is removed and replacedwith compounds diluted in culture medium. Six serial two-fold dilutionsof drugs are tested in triplicate. After further 72-h incubation, avolume of 50 μL of a 10 μCi/mL solution of [3H] methyl thymidine(Amersham Life Science, Inc., Arlington Heights, Ill.; 2 Ci/mmol) inculture medium is added and the plates are incubated for a further a 18h at 37° C. Cells are then washed with phosphate-buffered saline (PBS),trypsinized for 2 min, and collected onto a fiberglass filter using aTomtec cell harvester (Tomtec, Orange, Conn.). Filters are dried at 37°C. for 1 h and placed into a bag with 4.5 mL of liquid scintillationcocktail (Wallac Oy, Turku, Finland). The accumulation of [3H] methylthymidine, representing viable replicating cells, is measured using aliquid scintillation counter (1450-Microbeta; Wallac Oy). Ref. SOP:265-162-03. For this experiment, the cell lines used are; Huh-7 ET(cells derived from the Huh-7 cell line (hepatocellular carcinoma,human) and containing a HCV sub-genomic replicon), Molt-4 (peripheralblood, acute lymphoblastic leukemia, human), DU-145 (prostate carcinoma,metastasis to brain, human), Hep-G2 (hepatocellular carcinoma, human),and SH-SY5Y (neuroblastoma, human) cells.

Data Analysis

The 50% cytotoxic concentrations (CC50s) for cell toxicity aredetermined from dose response curves using six to eight concentrationsper compound in triplicate. Curves are fitted to data points usingnon-linear regression analysis, and IC50 values are interpolated fromthe resulting curve using GraphPad Prism software, version 2.0 (GraphPadSoftware Inc., San Diego, Calif., USA).

TI: Ratio of CC50/1050 in HCV replicon cells (Huh-7 ET cells) fromexample 12. When the compounds are tested more than once, the average ofthe TI is provided.

Table 2 lists the therapeutic indexes (Tis) of selected compoundsrepresentative of the invention relative to other thiophene HCVpolymerase inhibitors.

TABLE 2 Compound TI 1 ++++++ 2 +++ 3 ++++++ 4 ++++ 5 ++++++ 6 ++++++ 7+++++ 8 ++++++ 9 ++++++ A ++ B ++ C + D ++++ E +++ F ++ G +++ H + I ++TI +: <100 ++: 100-1000 +++: 1000-2000 ++++: 2000-4000 +++++: 4000-6000++++++: >6000

A

B

C

D

E

F

G

H

I

Compounds A to I can be synthesized as described in U.S. Pat. No.6,881,741, WO02/100851, WO2004/052885, or WO 2006/072347.

EXAMPLE 13 Stability in Human and Rat Microsomes and Induction inCultured Human Hepatocytes

Certain compounds according to the present invention and having aspecific substitution pattern, exhibit an improved microsome stabilityand/or Induction in human hepatocytes relative to other thiopheneanalogues. For example, compounds 1 and 2 showed better microsomestability profile and induction profile relative to compound E.

Stability in Human and Rat Microsomes

Each compound is incubated in hepatic microsomes (1.6 mg/mL) at 37°under oxidative and glucuronidation conditions (1.5 mM NADPH and 1.5 mMUDPGA in phosphate buffer, pH=7.4). Incubations containing no NADPH andno UDPGA are used as controls. The compounds are incubated at 50 uM for0 and 60 minutes. The reaction is stopped by the addition of an equalvolume of acetonitrile. The mixture is centrifuged and the supernatantis analyzed by HPLC/UV or MS/MS. The percentage parent remainingcorresponds to the area of the parent compound in the 60-minuteincubation versus the area of the parent compound in a 0-minuteincubation ×100.

Induction in Cultured Human Hepatocytes

The induction is performed in cryopreserved or fresh cultured-humanhepatocytes. The cells are cultured on collagen for 48 hours. Followingthis period, the cells are dosed with freshly spiked incubation mediacontaining the test compound or the positive control inducer,rifampicin. Final concentrations of the test compounds in theincubations are 1, 10 and 100 mM while the positive control is tested at10 mM. Negative controls (NC) consisted of incubating the cells with0.1% DMSO final content. The cell treatment is pursued for a total of 48hours with fresh incubation media spiked with test compound or controlinducer replaced daily. At the end of the induction period, the mediacontaining the inducer is removed and cells are washed twice with 200 uLKrebs-Henseleit buffer containing 12 mM HEPES, pH 7.4 (KH buffer). Theinduction of CYP3A4 is then measured by activity using testosterone assubstrate and by mRNA levels. For CYP3A4 activity, fresh KH buffer,spiked with the 200 mM testosterone is added and the cells are incubatedfor 30 minutes at 37° C. At the end of the incubation period, the mediais removed and analyzed by HPLC/MS for 6-beta-hydroxy testosteronedetermination. The maximal induction (100% induction) is determined withthe 10 mM Rifampicin treatment. The potential for test compounds tocause CYP3A Induction is described as a percentage of maximal inductionobtained with the classic inducer. For mRNA level determination,hepatocytes are harvested and total RNA is prepared using Qiagen RNeasyPurification Kit (Mississauga, ON) according to manufacturer'sinstructions. The cDNAs of the hepatocytes are synthesized from totalRNA using M-MLV Reverse Transcriptase (Invitrogen, Carlbad, Calif.) withuniversal primer (Roche Diagnostic, Germany). Analysis of specific mRNAexpression in total RNA samples is performed by quantitative real timePCR using Applied Biosystem's ABI Prism 7700 Sequence Detection System.Primers used for CYP3A4 are 5′-TCA GCC TGG TGC TCC TCT ATC TAT-3′ asforward primer and 5′-AAG CCC TTA TGG TAG GAC AAA ATA TTT-3′ as reverseprimer. The probe used was 5′-/56-FAM/TCC AGG GCC CAC ACC TCT GCCT/36-TAMSp/-3′. The data are normalized to Ribosomal 18S mRNA (VIC) withreal time detection. Data analysis and statistical tests are performedusing MicrosoftÒ Excel. Results can be reported as fold change geneexpression compared to control group according to the following formula:ΔCt=CtFAM−CtVICΔΔCt=ΔCt Drug Candidate−ΔCt ControlFold Induction:2−ΔΔCt

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention and, withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

We claim:
 1. A compound of formula IA:

wherein, R¹ is C₁₋₆ alkyl or C₃₋₆ cycloalkyl; X is

M is

R⁵ is cyclohexyl which is unsubstituted or substituted one or more timesby R¹³; R⁶ is

 or cyclohexyl which is substituted in the 4 position by R¹⁴; Y isCOOR⁷, COCOOR⁷, P(O)OR^(a)OR^(b), S(O)OR⁷, S(O)₂OR⁷, tetrazole,CON(R⁷)CH(R⁷)COOR⁷, CONR⁸R⁹, CON(R⁷)—SO₂—R⁷, CONR⁷OH and halogen; R⁷, R⁸and R⁹ are each independently H, C₁₋₁₂ alkyl which is unsubstituted orsubstituted one or more times by R¹⁰, C₂₋₁₂ alkenyl which isunsubstituted or substituted one or more times by R¹⁰, C₂₋₁₂ alkynylwhich is unsubstituted or substituted one or more times by R¹⁰, C₆₋₁₄aryl which is unsubstituted or substituted one or more times by R¹¹,C₇₋₁₆ aralkyl which is unsubstituted or substituted one or more times byR¹¹, 5-12 member heteroaryl which is unsubstituted or substituted one ormore times by R¹¹, 6-18 member heteroaralkyl which is unsubstituted orsubstituted one or more times by R¹¹, 3-12 member heterocycle which isunsubstituted or substituted one or more times by R¹², or 4-18 memberheterocycle-alkyl which is unsubstituted or substituted one or moretimes by R¹², or R⁸ and R⁹ are taken together with the nitrogen atom toform a 3 to 10 member heterocycle which is unsubstituted or substitutedone or more times by R¹² or a 5-12 member heteroaryl which isunsubstituted or substituted one or more times by R¹¹; and R_(a) andR_(b) are each independently chosen from H, C₁₋₁₂ alkyl which isunsubstituted or substituted one or more times by R¹⁰, C₂₋₁₂ alkenylwhich is unsubstituted or substituted one or more times by R¹⁰, C₂₋₁₂alkynyl which is unsubstituted or substituted one or more times by R¹⁰,C₆₋₁₄ aryl which is unsubstituted or substituted one or more times byR¹¹, C₇₋₁₆ aralkyl which is unsubstituted or substituted one or moretimes by R¹¹, 5-12 member heteroaryl which is unsubstituted orsubstituted one or more times by R¹¹, 6-18 member heteroaralkyl which isunsubstituted or substituted one or more times by R¹¹, 3-12 memberheterocycle which is unsubstituted or substituted one or more times byR¹², or 4-18 member heterocycle-alkyl which is unsubstituted orsubstituted one or more times by R¹², or R^(a) and R^(b) are takentogether with the oxygen atoms to form a 5 to 10 member heterocyclewhich is unsubstituted or substituted one or more times by R¹⁰ or a 5-12member heteroaryl which is unsubstituted or substituted one or moretimes by R¹¹; R¹⁰ is halogen, oxo, —NH₂, —NH(C₁₋₄ alkyl), —N(C₁₋₄alkyl)₂, —CONH₂, —CONH(C₁₋₄ alkyl), —CON(C₁₋₄ alkyl)₂, —NHCOH, —N(C₁₋₄alkyl)COH, —N(C₁₋₄ alkyl)COC₁₋₄ alkyl, —NHCOC₁₋₄ alkyl, —C(O)H,—C(O)C₁₋₄ alkyl, carboxy, —C(O)OC₁₋₄ alkyl, hydroxyl, C₁₋₄ alkoxy,nitro, nitroso, azido, cyano, —S(O)₀₋₂ H, —S(O)₀₋₂C₁₋₄ alkyl, —SO₂NH₂,—SO₂NH(C₁₋₄ alkyl), —SO₂N(C₁₋₄ alkyl)₂, —NHSO₂H, —N(C₁₋₄ alkyl)SO₂H,—N(C₁₋₄ alkyl)SO₂C₁₋₄ alkyl, or —NHSO₂C₁₋₄ alkyl; R¹¹ is halogen, C₁₋₆alkyl, halogenated C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ alkoxy,—NH₂, —NH(C₁₋₄ alkyl), —N(C₁₋₄ alkyl)₂, —CONH₂, —CONH(C₁₋₄ alkyl),—CON(C₁₋₄ alkyl)₂, —NHCOH, —N(C₁₋₄ alkyl)COH, —N(C₁₋₄ alkyl)COC₁₋₄alkyl, —NHCOC₁₋₄ alkyl, —C(O)H, —C(O)C₁₋₄ alkyl, carboxy, —C(O)OC₁₋₄alkyl, hydroxyl, C₁₋₆ alkoxy, nitro, nitroso, azido, cyano, —S(O)₀₋₂H,—S(O)₀₋₂C₁₋₄ alkyl, —SO₂NH₂, —SO₂NH(C₁₋₄ alkyl), —SO₂N(C₁₋₄ alkyl)₂,—NHSO₂H, —N(C₁₋₄ alkyl)SO₂H, —N(C₁₋₄ alkyl)SO₂C₁₋₄ alkyl, or —NHSO₂C₁₋₄alkyl; R¹² is halogen, oxo, C₁₋₆ alkyl, halogenated C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, —NH₂, —NH(C₁₋₄ alkyl), —N(C₁₋₄ alkyl)₂, —CONH₂,—CONH(C₁₋₄ alkyl), —CON(C₁₋₄ alkyl)₂, —NHCOH, —N(C₁₋₄ alkyl)COH, —N(C₁₋₄alkyl)COC₁₋₄ alkyl, —NHCOC₁₋₄ alkyl, —C(O)H, —C(O)C₁₋₄ alkyl, carboxy,—C(O)OC₁₋₄ alkyl, hydroxyl, C₁₋₆ alkoxy, nitro, nitroso, azido, cyano,—S(O)₀₋₂H, —S(O)₀₋₂C₁₋₄ alkyl, —SO₂NH₂, —SO₂NH(C₁₋₄ alkyl), —SO₂N(C₁₋₄alkyl)₂, —NHSO₂H, —N(C₁₋₄ alkyl)SO₂H, —N(C₁₋₄ alkyl)SO₂C₁₋₄ alkyl, or—NHSO₂C₁₋₄ alkyl; R¹³ is OH, halogen, C₁₋₆-alkyl, halogenatedC₁₋₆-alkyl, C₁₋₆-alkoxy, halogenated C₁₋₆-alkoxy, cyano, nitro, —NH₂,—NH(C₁₋₄ alkyl), —N(C₁₋₄ alkyl)₂, —CONH₂, —CONH(C₁₋₄ alkyl), —CON(C₁₋₄alkyl)₂, —NHCOH, —N(C₁₋₄ alkyl)COH, —N(C₁₋₄ alkyl)COC₁₋₄ alkyl,—NHCOC₁₋₄ alkyl, —C(O)H, —C(O)C₁₋₄ alkyl, carboxy, —C(O)OC₁₋₄ alkyl,—S(O)₀₋₂C₁₋₄ alkyl, —SO₂NH₂, —SO₂NH(C₁₋₄ alkyl), —SO₂N(C₁₋₄ alkyl)₂,—N(C₁₋₄ alkyl)SO₂H, —N(C₁₋₄ alkyl)SO₂C₁₋₄ alkyl, —NHSO₂C₁₋₄ alkyl,C₆₋₁₄-aryl, C₆₋₁₄-aryloxy, or C₆₋₁₄-aryloxy-C₁₋₆-alkyl,; R¹⁴ is OH,halogen, C₁₋₆-alkoxy, C₁₋₆-alkyl, C₁₋₆-alkyl-CO—NH—,C₁₋₆-alkyl-CO—N(C₁₋₆-alkyl)-, or heteroaryl; and R^(14a) is C₁₋₆-alkyl,C₃₋₇-cycloalkyl, halogenated C₁₋₆-alkyl, C₁₋₆-alkyl-CO—, —S(O)₀₋₂C₁₋₄alkyl, heteroaryl or C₆₋₁₄-aryl; or a pharmaceutically acceptable saltthereof.
 2. A compound according to claim 1, wherein Y is COOR⁷ and R⁷is H, methyl, or ethyl.
 3. A compound according to claim 2, wherein Y isCOOR⁷ and R⁷ is H.
 4. A compound according to claim 1, wherein R¹⁴ isOH, fluoro, C₁₋₆-alkoxy, or triazole.
 5. A compound according to claim4, wherein R¹⁴ is OH, —OCH₃, 1,2,3 triazole or 1,2,4 triazole.
 6. Acompound according to claim 1, wherein R¹ is C₁₋₆ alkyl orC₃₋₆cycloalkyl which are unsubstituted or substituted one or more timesby —NH₂, NHCH₃, N(CH₃)₂, or hydroxyl.
 7. A compound according to claim6, wherein R¹ is C₁₋₆ alkyl or C₃₋₆cycloalkyl.
 8. A compound accordingto claim 4, wherein R¹ is methyl, ethyl, propyl, isopropyl, butyl,sec.-butyl, tert.-butyl, cyclopropyl, cyclobutyl, cyclopentyl, orcyclohexyl.
 9. A compound according to claim 6, wherein R¹ is methyl,ethyl, propyl, isopropyl, butyl, sec.-butyl or tert.-butyl.
 10. Acompound according to claim 1, wherein X is —NR⁶—CO—R⁵ and R⁵ iscyclohexyl which is unsubstituted or substituted in the 4-position byOH, halogen, C₁₋₆-alkyl, halogenated C₁₋₆-alkyl, C₁₋₆-alkoxy,halogenated C₁₋₆-alkoxy, cyano, nitro, —NH₂, —NH(C₁₋₄ alkyl), or —N(C₁₋₄alkyl)₂, wherein the 4-position substituent is in the trans positionrelative to the carbonyl group.
 11. A compound according to claims 10,wherein R⁵ is cyclohexyl which is unsubstituted or substituted one ormore times by OH, halogen, C₁₋₆-alkyl, halogenated C₁₋₆-alkyl,C₁₋₆-alkoxy, halogenated C₁₋₆-alkoxy, cyano, nitro, —NH₂, —NH(C₁₋₄alkyl), or —N(C₁₋₄ alkyl)₂.
 12. A compound according to claim 10,wherein R⁵ is cyclohexyl which is substituted in the 4-position one ormore times by OH, halogen, C₁₋₆-alkyl, halogenated C₁₋₆-alkyl,C₁₋₆-alkoxy, halogenated C₁₋₆-alkoxy, cyano, nitro, —NH₂, —NH(C₁₋₄alkyl), or —N(C₁₋₄ alkyl)₂.
 13. A compound according to claim 10,wherein R⁵ is cyclohexyl which is substituted in the 4-position byC₁₋₆-alkyl.
 14. A compound according to claim 10, wherein R⁵ iscyclohexyl which is substituted in the 4-position by —CH₃.
 15. Acompound according to claim 1, wherein R⁶ is cyclohexyl which issubstituted one or more times by OH, halogen, C₁₋₆-alkyl, orC₁₋₆-alkoxy.
 16. A compound according to claim 15, wherein R⁶ iscyclohexyl which is substituted in the 4-position one or more times byOH, halogen, C₁₋₆-alkyl, or C₁₋₆-alkoxy.
 17. A compound according toclaim 15, wherein R⁶ is cyclohexyl which is substituted in the4-position and the 4-position substituent is in the trans positionrelative to the amino group.
 18. A compound according to claim 15,wherein R⁶ is cyclohexyl which is substituted in the 4-position by OH orC₁₋₆-alkoxy.
 19. A compound according to claim 15, wherein R⁶ iscyclohexyl which is substituted in the 4-position by OH or methoxy. 20.A compound according to claim 1, wherein: R¹ is methyl, ethyl, propyl,isopropyl, butyl, sec.-butyl, tert.-butyl, cyclopropyl, cyclobutyl,cyclopentyl, or cyclohexyl; R⁵ is cyclohexyl which is unsubstituted orsubstituted one or more times by OH, C₁₋₄-alkyl, or C₁₋₄-alkoxy; R⁶ iscyclohexyl which is substituted by OH, F, C₁₋₄-alkoxy, C₁₋₄-alkyl, orhalogenated C₁₋₄-alkyl; Y is COOH.
 21. A pharmaceutical compositioncomprising at least one compound according to claim 1 and at least onepharmaceutically acceptable carrier or excipient.
 22. A pharmaceuticalcombination comprising at least one compound according to claim 1 and atleast one additional agent.
 23. A pharmaceutical combination accordingto claim 22, wherein said at least one additional agent is selected fromviral serine protease inhibitors, viral polymerase inhibitors, viralhelicase inhibitors, immunomudulating agents, antioxidant agents,antibacterial agents, therapeutic vaccines, hepatoprotectant agents,antisense agents, inhibitors of HCV NS2/3 protease and inhibitors ofinternal ribosome entry site (IRES).
 24. A pharmaceutical combinationaccording to claim 22, wherein said at least one additional agent isselected from ribavirin and interferon-α.
 25. A pharmaceuticalcombination according to claim 22, wherein said at least one additionalagent is selected from ribavirin and pegylated interferon-α.
 26. Amethod for treating a Hepatitis C viral infection in a host comprisingadministering to the host a therapeutically effective amount of at leastone compound according to claim
 1. 27. A method according to claim 26,further comprising administering at least one additional agent.
 28. Amethod according to claim 26, wherein said at least one additional agentis selected from viral serine protease inhibitors, viral polymeraseinhibitors, viral helicase inhibitors, immunomudulating agents,antioxidant agents, antibacterial agents, therapeutic vaccines,hepatoprotectant agents, antisense agents, inhibitors of HCV NS2/3protease and inhibitors of internal ribosome entry site (IRES).
 29. Themethod according to claim 28, wherein said at least one additional agentis selected from ribavirin and interferon-α.
 30. The method according toclaim 28, wherein said at least one additional agent is selected fromribavirin and pegylated interferon-α.